Critical Reviews in Biotechnology

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Astaxanthin from Microbial Sources

Eric A. Johnson & Gil-Hwan An

To cite this article: Eric A. Johnson & Gil-Hwan An (1991) Astaxanthin from Microbial Sources,
Critical Reviews in Biotechnology, 11:4, 297-326, DOI: 10.3109/07388559109040622

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 Critical Reviews in Biotechnology, 11(4):297-326 (1991)

Astaxanthin from Microbial Sources

Eric A. Johnson
Department of Food Microbiology and Toxicology, University of Wisconsin, Madison, Wisconsin

Gil-Hwan An
Hai-Tai Food Research Institute, Hai-Tai Confectionery Co., Yungdeungpo-Goo, Seoul, South
Korea

ABSTRACT: Salmon farming increased substantially in the 1980s, which created a large market for astaxanthin,
the principal pigment of salmon. Carotenoids are the most widely distributed class of pigments in nature and
have essential biological functions in animals. They also impart attractive pigmentation to many farmed animals.
Since animals lack the ability to synthesize carotenoids, the pigments must be supplemented to feeds, usually
at considerable expense to the farmer (10 to 15% of total feed costs). Astaxanthin (3,3’-dihydroxy+,p-carotene-
4,4’-dione) is the principal carotenoid pigment of salmonids and gives attractive pigmentation in the eggs, flesh,
and skin. Worldwide production of farm-raised salmon increased rapidly in the past decade, and more than
200,000 T were raised in 1990. Currently, chemically synthesized astaxanthin and canthaxanthin (p,p-carotene-
4,4’-dione) are added to salmonid feeds as pigmenters, but there is considerable interest within the aquaculture
industry in using natural sources of astaxanthin. The principal biological sources being considered are crustacea
and crustacean extracts, the green microalga Haematococcus, and the yeast Phafla rhodozyma. Each natural
pigment source has its limitations and they currently cannot compete economically with the synthetic additive.
Crustacean meals have relatively low contents of astaxanthin and high levels of moisture, ash, and chitin. The
alga Haematococcus has a high concentration of astaxanthin (0.2 to 2%), but industrial application is limited
by the lengthy autotrophic cultivation in open freshwater ponds and the requirement for disruption of the cell
wall to liberate the carotenoid. The yeast P . rhodozyma has desirable properties as a biological source of pigment,
including rapid heterotrophic metabolism and production of high cell densities in fermentors, but its content of
astaxanthin in wild strains is only 200 to 300 pg/g yeast (0.02 to 0.03%). Mutants have been isolated that
produce >3000 pg total carotenoid per gram of yeast (>0.30%) in shake flasks after 5 d growth, and measurement
of carotenoid fluorescence in individual cells indicates that levels of 10,000 to 15,000 pg/g can be obtained.
High-producers, however, are often unstable and further strain development is required. In this article, biological
sources of astaxanthin are critically evaluated and compared with the synthetic compound presently being used
in animal feeds.

KEY WORDS: astaxanthin, carotenoids, Phafla rhodozyma, salmon, pigmentation.

1. INTRODUCTION red coloration to the animals and contributes to

consumer appeal in the marketplace. Carotenoids
Astaxanthin (3,3’-dihydroxy-P,P,-carotene- also have important metabolic functions in ani-
4,4’-dione) is widely distributed in nature and is mals and man, including conversion to vitamin
the principal pigment in crustaceans and salmo- A, enhancement of the immune response, and
nids, various birds including certain flamin- protection against diseases such as cancer by
goes and the scarlet ibis, and many other organ- scavenging of oxygen radical^.^.' Since animals
isms. The carotenoid imparts distinctive orange- cannot synthesize carotenoids, the pigments must

0738-8551/91/$.50
0 1991 by CRC Press, Inc.

297
be supplemented in the feeds of farmed spe- but feeding studies have shown that astaxanthin
cies. 1.2.4-6 (3,3’-dihydroxy-P, p ,-carotene-4,4.-dione; Fig-
Fish farming currently provides 10 to 15% ure 2) is superior as a pigmenter.6 Astaxanthin
of the seafood consumed in the world8 and salmon is also the carotenoid found naturally in salmo-
is one of the most important aquacultured prod- nids, 1.5.1 1-14 and is the preferred pigment in aqua-
u c t ~Production
.~ of farmed salmon has increased culture since it is deposited more efficiently than
dramatically in the past decade (Figure 1) and other carotenoids in salmon and trout.6 Astax-
>200,000 T were produced worldwide in 1989, anthin occurs in salmonids at levels ranging from
primarily in N o ~ w a yPresently,
.~ three out of every 3 to 37 mg/kg6 Most fish farmers are using syn-
ten salmon consumed in the world are raised in thetic astaxanthin and canthaxanthin in feeds at
pens. By the year 2000 it is expected that farmed levels of 35 to 75 mg/kg dry feed,6 but the syn-
salmon will dominate the markets for Atlantic, thetics are expensive (astaxanthin costs $2000 to
coho, and chinook salmon, and that Canada, $2500 (U.S.)/kg), and have not been approved
Chile, and Japan will emerge as major producers by the U.S. Food and Drug Administration (FDA)
of Pacific ~ a l m o nRecent
.~ forecasts are that total for use in salmonid rations. l5 Furthermore, the
production of farmed salmon will be 286,000 and synthetic formulations may contain unnatural
460,000 T i n 1990 and 2000, re~pectively.~ Trout configurational and cis-trans-isomers and caro-
is also farmed worldwide and >250,000 T were tenoid-like compounds in the preparations. “3”

-
produced in 1987.1° The attractiveness and mar-
ket value of trout can also be enhanced by
pigmentation.6

200 %Carotene

-isf
zE 0
Canthaxanthin

L
E 100
0

L.
0
M
C
0
c1
W
.I
HO Astaxanthin
0

1980 1982 1984 1986 1988 1990

0
Year

FIGURE 1. Production of farmed salmon, 1981 to FIGURE 2. Chemical structures of carotenoids im-
1 988.9 portant in aquaculture.

The growth of salmonid aquaculture in the There is also a trend toward using natural sources
past decade has created a need for carotenoids of feed nutrients and an increasing wariness by
for trout and salmon feeds.6 In the early 1980s, the farmer and consumer of introduction of syn-
synthetic canthaxanthin (p, P-carotene-4,4’-dione) thetic chemicals in the food chain, even though
stabilized in beadlets was the preferred pigment, the chemicals may be identical to naturally oc-
curring compounds. All these factors have con- solvents but can be dissolved at room temperature
tributed to interest in natural sources of caro- in dichloromethane (-30 gA), chloroform (- 10
tenoids. g/l), acetone (-0.2 g/l), dimethylsulfoxide
The provision of pigments in feeds of farmed (DMSO) (-0.5 g/l), and other nonpolar solvents.
salmon is an expensive practice - astaxanthin Its absorption spectrum represents a conjugated
is one of the most costly components of salmon polyene structure,,,A = 489 nm in chloroform,
feeds, accounting for 10 to 15% of total feed 478 nm in ethanol, and 480 nm in a c e t ~ n e . ~ ~ , ~ ~ , ~ ~
costs.6 On a dry basis, astaxanthin currently is Methods have been comprehensively de-
sold for > $2000/kg (marketed as CarophyllB scribed for the analysis of c a r ~ t e n o i d s . Be-
~~,~~
pink, Hoffmann-LaRoche, Inc., Basel, Switz- cause carotenoids contain a long conjugated dou-
erland, containing a minimum of 8% astaxanthin ble bond system, they are less stable than other
per kilogram). Torrissen et a1.6 reported that isoprenoids and precautions must be taken to avoid
>6000 kg of carotenoid pigments were used in artifacts and destruction of the p i g r n e n t ~ . ~ ~ , ~ ~
the feeds of farmed salmonids in 1986. Based on Light, heat, acids, and oxygen are particularly
current production trends, it is expected that detrimental to carotenoids, and enzymatic de-
>15,000 kg will be required by 1990, and pos- struction also can occur during extraction from
sibly >100,000 kg by the year 2000. This po- biological samples. High-pressure liquid chro-
tential demand could open a large market for matography (HPLC) is useful for detecting cis-
microbially produced astaxanthin. Several com- isomers of astaxanthin, which generally elute later
panies are investigating production of natural from the column. All-trans natural astaxanthin
sources of astaxanthin. Presently, the most prom- and other carotenoids are readily isomerized to
ising recognized natural sources of astaxanthin cis-trans mixtures, especially the 9 4 s and 13-
are crustacea (mainly the shrimp Pandalus bo- cis unhindered isomers, and precautions must be
realis and the krill Euphanasia pacifica, E . su- taken to avoid their formation.
29730 cis-Isomers of
perba), the microalga Haernotococcus, and the carotenoids are generally considered to be un-
yeast Phafla rhodozyma. Since the authors’ study natural artifacts. However, cis-trans isomerism
P. rhodoqrna, this article focuses on PhafSia as of vitamin A (retinol) occurs naturally in the ret-
a biological source of astaxanthin. ina, and the 11-cis-retinol isomer is thermodyn-
amically less stable and energy is required for
the endergonic transformation of vitamin A to
II. CHEMICAL PROPERTIES OF 1I-cis-retin~l.~~Hence, it is possible that cis-
ASTAXANTHIN isomers of carotenoids may have essential met-
abolic functions and are naturally formed in cer-
Astaxanthin (3,3’-dihydroxy-P,P-carotene- tain organisms. Our laboratory routinely detects
4,4’-dione) (Figure 2)18-20is an oxycarotenoid cis-astaxanthin on analysis of carotenoids in P.
with the molecular formula C,,,H5,O4 and has a rhodozyrna. During analysis of astaxanthin and
mol wt of 596.86.21.22Astaxanthin was first its esters from natural sources, alkaline condi-
chemically identified by Kuhn and Sorensen18 tions should be avoided since irreversible con-
based on Karrer’s earlier recognition of astacene, version to astacene (Figure 2) takes place in alkali
and Davis and WeedonZ3 later confirmed the and oxygen and on saponification. A specialized
structure by partial synthesis of its derivative as- anaerobic procedure has been described to min-
tacene from canthaxanthin. The total synthesis imize conversion to a ~ t a c e n e Carotenoids
.~~ are
of optically pure astaxanthin from racemic inter- generally extracted with water-miscible polar or-
mediates has also been ac~omplished,*~-~~ mainly ganic solvents, but chloroforrdmethanol, which
by researchers at F. Hoffmann-LaRoche, Basel, is used extensively for extraction of other lipids,
Switzerland. is not recommended since it may contain traces
Isolated crystalline astaxanthin has the ap- of hydrochloric acid. Mechanical disruption of
pearance of a fine, dark violet-brown powder. biological samples is usually necessary to achieve
Its melting point is approximately 224°C. It is quantitative extraction, although hot DMSO
insoluble in aqueous solutions and most organic has been used successfully for extraction of

299
P. r h ~ d o z y m a .Following
~~ extraction, carot-
enoids can be separated conveniently by thin-
layer chromatography (TLC) and HPLC.30Spec-
trophotometry is used for quantitative determi-
no
nation of astaxanthin. For most carotenoid de- 0
terminations the specific absorption coefficient
[A(1%/1 cm) = E( 1%/ 1 cm)] is used to calculate
the concentration. It is strongly recommended
that pure carotenoids be used as standards during
analysis. Unfortunately, pure crystalline astax- no
0
anthin for use as standard is not readily available
from commercial chemical suppliers. Hence, 0
OH
various methods and absorbency coefficients have
been used for the analysis of astaxanthin, which
has resulted in discrepancies among laboratories
in quantities reported in biological samples. F. (3S,S’R-meso)
0
Hoffmann-La Roche Ltd. (personal communi-
cation) recommends that a standard solution of FIGURE 3. Structures of configurational isomers of
astaxanthin (1 to 2 kg/ml) be prepared in hexane, astaxanthin.
having a maximum absorbancy at 472 nm and a
standard absorbency [E (1%/1 cm)] of 1910 of a
1% astaxanthin solution (w/v). The purity of the dently supported that the 3s,3’S configurational
standard should be checked by HPLC on a silica isomer occurred predominately in Homarus. The
column pretreated with phosphoric acid and de- predominant configurational isomer in Haema-
tected by absorption at 470 nm. For analysis of tococcus pluvialis was shown to be 3S,3’S.36In
P. rhodozyma , our laboratory currently extracts 1975 Andrewes and St&’ found that the type
with hot DMSO in the useful assay developed strain of Ph$ia rhodozym contained the 3R,3’R
by Sedmak et al. 33 and an E (1%/ 1 cm) in acetone form as its predominant (92%) configurational
of 2100. Extraction with DMSO is much less isomer. At the time, PhaSfia was thought to con-
tedious than methods using mechanical or en- tain astaxanthin of the opposite configuration
zymatic disruptions of cells. commonly found in nature. Industry was reluc-
Astaxanthin has two asymmetric carbon at- tant to develop the yeast commercially since it
oms at the 3 and 3’ positions and can exist in was not known if the FDA would consider the
four configurations, including the identical en- R,R’ isomer to be unnatural and prohibit its ad-
antiomers (3S,3’S; 3R,3‘R) and meso forms dition to feeds and foods. Since the early studies
(3R,3’S; 3’R,3S) (Figure 3). Chemical synthesis of Andrewes and collaborators, several investi-
from racemic precursors gives equal mixtures of gators have detected both meso and enantiomeric
the configurational isomers. The isomers can be forms of both configurational isomers from var-
separated by reacting ( - )-camphanic diastereo- ious natural sources, including lobster eggs
meric diesters by HPLC and by TLC on silica (Homarus gammarus), shrimp (Pandalus bo-
gel.34 cis-trans-Isomers of the configurational realis), wild salmon, and ~ m p l a n k t o n . Maoka
~~-~~
isomers also can be separated by HPLC.32*34 et al.42recently reported that (3R,3’R) astaxan-
Elegant chemical research has elucidated the thin was the predominant configurational isomer
configurational isomers present in natural sources in the Antarctic krill, Euphanasia superba. All
of astaxanthin. The 3 s ,3’S configuration from meso and enantiomeric configurational isomers
lobster (Homarus gammarus) eggs and Haema- of astaxanthin have been detected in wild
tococcus pluvialis was demonstrated by An- salmon,4o and it is presently believed that the
drewes et al. in 1974.20X-ray a n a l ~ s i s ~ of
~.~’ Occurrence of particular isomers reflects the source
astaxanthin synthesized from (4R,6R)-4-hy- of the food in nature and is not relevant biolog-
droxy-2,2,2-trimethylcyclohexanone indepen- ically in the salmon.

300
111. CAROTENOID PIGMENTATION OF be deposited equally well in salmonids. When
SALMONIDS racemic astaxanthin was fed to salmon or trout,
the proportion of isomers detected in the flesh
Astacene (3,3 '-dihydrox y-2,3,2' ,3 ‘-tetrade- was identical to that present in the feed.6J7*43,48
hydro-P,P-carotene-4,4’-dione; Figure 2) was re- During reductive metabolism of astaxanthin to
ported in the early 1900s as the principal caro- zeaxanthin, epimerization from 3 s to 3R isomer
tenoid in salmon id^.^ Later it was found that was reported. Rainbow trout appeared to
17946*47

astacene is a degradation product of naturally preferentially hydrolyze the R configurational

occurring astaxanthin. The astaxanthin content isomers of the palmitate esters, suggesting that
of salmon varies considerably depending on spe- the esterases have stereochemical preference for
cies, sex, nutrition, maturity, and health.6Mature the R configuration. It was observed by Katsuy-
Atlantic salmon contain 3 to 8 ppm, while sock- ama et al.49that the (3R,3’R) astaxanthin diester
eye salmon can have contents as high as 37 ppm was deposited in the flesh of rainbow trout two
imparting a vivid pigmentation. In nature, inges- times more efficiently than the (3R,3’S) and four
tion of zooplankton probably leads to flesh pig- times more efficiently than the (3S,3’S) diesters,
mentation.6Astaxanthin occurs mainly in the free but further confirmation of this observation is
form (unesterified) in salmonid flesh, and ester- needed.
ified in the skin and ovaries.6 Astaxanthin levels
of 3 to 4 mg or above have been reported to give
a satisfactory visual impression of the fish flesh;6 IV. PIGMENT SOURCES FOR FARMED
however, carotenoids can fade or isomerize dur- SALMON
ing handling and it is recommended that levels
>4 mg/kg be reached in salmon sent to market.6 A. Synthetic Astaxanthin and
The uptake of astaxanthin and other carot- Canthaxanthin
enoids from the feed depends on the chemical
structure of carotenoid, the proportion of cis-iso- F. Hoffmann-La Roche, Basel, Switzerland,
mers, esterification, and association of the car- have developed over many years advanced
otenoid with fats or proteins. Carotenoids are syntheses for various carotenoids. In 1964 they
generally absorbed with poor efficiency by ani- introduced canthaxanthin as a pigmenter for foods
mals (Q10% of carotenoid in the feed) and little and feeds (“Roxanthin” or “Carophyll red’’).50
is known of the uptake mechanism and of the Recently Roche accomplished the synthesis of
means for enhancing uptake. Astaxanthin occurs trans-astaxanthin, which is marketed as “Caro-
predominately as esters in krill“ and the mi- phyll pink”. Synthetic astaxanthin is presently
croalga Haematococcus ,36 but occurs freely in the principal source used in feeds. Astaxanthin
Phafia r h ~ d o z y m a .Free
~ ~ astaxanthin was re- is preferred as a pigmenter over synthetic can-
ported to be more efficiently deposited in fish thaxanthin for salmonids since it is more effi-
muscle than esterified astaxanthin and canthax- ciently absorbed and also imparts a more natural
anthin,6.46*47although this has been disputed in color to salmon.6 Chemically synthesized astax-
feeding trials using krill astaxanthin diester and anthin sells for $2000 to $2500/kg (dry weight
synthetic free a~taxanthin.~~ It is possible that the basis) in beadlets containing 5% astaxanthin and
krill astaxanthin was more efficiently deposited is stabilized by various ingredients, including
in salmonids than the synthetic due to association gelatin, sucrose, corn starch, modified food
with lipids or other compounds promoting ab- starch, ascorbyl palmitate, and e t h o x y q ~ i nThe
.~~
sorption, but further studies are required to un- cis-astaxanthin content is not to exceed 2%.
derstand the mechanisms of uptake, metabolism, Analyses of commercial preparations of canthax-
and deposition. anthin by reversed-phase HPLC indicated that
Since astaxanthin has two identical chiral various isomers were present, including
centers, it exists as three configurational isomers all-trans-, all-cis-, and other isomers of can-
in nature: (3S,3’S), (3S,3’R), and (3R,3’R) (Fig- thaxanthin. l6 The ratio of all-trans to all-cis-can-
ure 3). Each configurational isomer appears to thaxanthin extracted from the beadlets was ap-

301
proximately 3: I . I 6 Analyses of independent lots and excluding the use of astacene. One process
of canthaxanthin also showed the presence of the involved the oxidation by a percarboxylic acid
four isomers, but these isomerizations may have of a silyl-enol ether that protected hydroxy groups.
occurred during handling following chemical Astaxanthin was obtained from canthaxanthin or
synthesis and release from Roche. precursors of canthaxanthin in three to five chem-
The commercial procedures used presently ical steps and in 67% yield.52Total syntheses of
in the chemical synthesis of astaxanthin are not astaxanthin have also been achieved in schemes
available to the general public and it is not pos- involving condensations of C, + c6 and C,, +
sible to accurately estimate the cost of manufac- C, to the CIS-phosphonium The actual
ture. F. Hoffmann-La Roche and others have commercial process used for chemical synthesis
published several methods of synthesis of astax- and production costs are not publicly available,
anthin and related xanthophylls To-
.25-28.32,35*51752 but precursor carotenoids (e.g., canthaxanthin)
tal synthesis of (3S,3'S)-astaxanthin and sell commercially for -$1200/kg.
(3R,3'R)-zeaxanthin has been achieved through
the preparation of optically active synthons using
chemical and microbiologically catalyzed reac- B. Crustaceans and Crustacea
tion steps. 5 1 The value of microbiological trans- Byproducts
formations includes the stereoselective modifi-
cation of specific carbons and oxygen atoms in Several researchers have evaluated crustacea
the carotenoid precursors. An important precur- and derivatives as pigment sources (reviewed in
sor for the synthesis of astaxanthin is (S)-3-ace- Tomssen et a1.6). In Norway, shrimp (Pundalus
toxy-4-oxo-P-ionone, which can be obtained by borealis) wastes have been used traditionally as
asymmetric hydrolysis of the (R)-terpene alcohol natural pigment sources for trout and salmone6
acetates by various micro~rganisms.~~ The ( S ) - Carotenoid levels in most crustacean prepara-
3-acetoxy-4-oxo-~-ionone is isolated in 78% the- tions, however, are usually quite low (0 to 200
oretical yield by extraction, countercurrent dis- mg/kg)6,54and satisfactory pigmentation requires
tribution, and recrystallization. The biotransfor- the addition of 10 to 25% by weight of the chi-
-
mation was improved 12-fold by selecting more tinous extract to the bulk diet. Crustacean wastes
active mutants after UV exposure and optimi- have high levels of ash, chitin, and moisture and
zation of fermentation conditions.28The precur- low levels of protein and other nutrients that limit
sor was converted to the C,, Wittig salt, which their usefulness.6Developments in extraction and
is coupled at both ends with the required C,, concentration of the carotenoids could improve
dialdehyde such that (3s,3'S)-astaxanthin is con- the feasibility of crustacean wastes for pigmen-
structed by the scheme e*,, + C*,, + C*,,.53 tation. Torrissen et al." concluded that shrimp
Hence, a combination of chemical and micro- meal has limited potential as an astaxanthin source
biologically catalyzed steps have been used suc- for salmonids.
cessfully in the selective industrial preparation of
chiral astaxanthin. Potential limitations of mi-
crobiologically catalyzed steps include low yields, C. Algae
poor toleration of substrates by the organisms,
and difficulty in recovering and preventing ex- Certain green algae in the subphylum Chto-
tensive conversion of intermediate^.^' rophyceae possess astaxanthin as their primary
Several approaches have also been used for c a r ~ t e n o i d Depending
. ~ ~ ~ ~ ~ on the method and
syntheses using carotenoid intermediates and also control of culture, very high levels accumulate
for total chemical syntheses." Early work showed in Huematococcus pluvialis (0.5 to 2% astax-
that it was possible to manufacture astaxanthin anthin on a dry weight b a s i ~ ) . ~Most
" ~ of the
from canthaxanthin in low yield via astacene and astaxanthin (87%) occurs esterified, which may
c r u ~ t a x a n t h i n .Bernhard
~ ~ , ~ ~ et al.27,52 developed affect its deposition and metabolism in some an-
a process for the chemical manufacture of astax- imals. Low deposition of astaxanthin by feeding
anthin using canthaxanthin or other intermediates algae to salmon was obtained by Kvalheim and

302
KnutSen,61and it was suggested that this was due (nine strains) and Alaska (one strain), Phaff et
to astaxanthin being present principally as esters; a1.62proposed the name Rhodozyma montanae,
however, poor deposition caused solely by es- describing a new genus and species. A Latin di-
terification was not confirmed by others.6 An- agnosis was not given as required by the Inter-
other possible limiting factor is availability of national Code of Botanical Nomenclature, which
carotenoids from the algal biomass. Highly pig- later allowed the change of the genus name to
mented algae occur in an encysted form sur- P h ~ f i a , "in~ honor of many years of research in
rounded by a thick cell wall, and this barrier yeast biology by Phaff. The yeast was described
could also impede the absorption of pigments. by Miller and associates as having several unique
characteristics, principally its fermentative ca-
pabilities, unusual carotenoid composition, and
D. Yeast cell wall composition. Other investigators have
found Phafia in nature. Golubev et a1.66isolated
The yeast Phaffia rhodozyma (Figure 4) was 67 strains from birch fluxes in the Moscow region
first isolated during the 1970s by Herman Phaff, of the U.S.S.R. The yeast has been isolated ex-
Martin Miller, Minoru Yoneyama, and Masumi clusively from broad-leaved trees in limited geo-
Soneda from exudates of deciduous trees in Ja- graphic regions of the
pan, Alaska, and the U.S.S.R.62-65 Phaff et a1.62 The genus Phafia is characterized by the
recognized that the yeast had several unusual synthesis of carotenoid pigments, production of
characteristics, and stated that the most remark- cell surface-associated amyloid compounds, a
able property was that the colonies were red to coenzyme Q-10 system, and the ability to ferment
orange due to the presence of carotenoid pig- sugars. Other pertinent properties include its abil-
ments, but at the same time the ten strains also ity to assimilate carbon compounds, including D-
fermented glucose, maltose, sucrose, and raffi- glucose, maltose, sucrose, cellobiose, trehalose,
nose. Since all of the strains were isolated from raffinose, soluble starch (latent or negative),
broad-leaved trees in mountainous areas of Japan ethanol (latent or negative), a-methylglucoside

flGURE 4. Scanning electron micrograph of Phaffia rhodozyma (strain 67-210) showing budding vegetative
cells, elongated cell with jagged collar bud scar, and spheroidal chlamydospore. (Magnification x 6500.)

303
(latent or negative), D-mannitol, salicin (weak), its mycelial phase and a teleomorph similar to
2-ketogluconate, DL-lactate (latent), succinate, that found in C. capitatum, and is probably not
and glycerol (weak). The yeast does not grow on closely related to the Trernellaceae, as originally
lactose, galactose, glucosamine, D-ribose, or D- suggested by Miller et al.63
arabinose and does not utilize nitrate, but does A major property that distinguishes Phafia
hydrolyze urea. It grows from 0 to 27°C. Its from other genera of related yeasts is the com-
mol% G + C is 48.3 * 0.18. The type strain is position of carotenoids of yeasts. Astaxanthin was
deposited in various yeast culture collections identified as the major pigment in P. rhodozyma
(UCD-FST#67-210; ATCC#24202; CBS 5905). by Andrewes working in the Starr laboratory at
Phafia is basidiomycetous in origin, but a the University of California at Davis.45 After
sexual cycle has not been demonstrated. Its bas- identifying astaxanthin as the major pigment, and
idiomycetous relationship was elucidated by the following up his work in Trondheim, Norway,
demonstration of a multilayered cell wall and on the absolute configuration of astaxanthin in
enteroblastic budding.63Its basidiomycetous af- lobster (Homarus), Andrewes determined the
finity is supported by the carbohydrate compo- configuration of yeast astaxanthin and made the
sition of the cell wall.67Phafia is phylogeneti- unexpected discovery that the predominant iso-
cally related to other carotenoid-forming imperfect mer was 3R,3'R,37 the opposite of his earlier
yeasts, including Rhodotorula, Cryptococcus, and finding of 3S,3'S in lobster." In his analysis of
other heterobasidiomycetous y e a s t ~ .Phafia
~~-~~ Phafia carotenoids, Andrewes also detected a
can be distinguished from the genus Rhodotorula new carotenoid, 3-hydroxy-3,4'-didehydro-P,Y-
by its production of amyloid compounds and by caroten-4-one (HDCO). Andrewes further noted
its ability to ferment sugars, and from Crypto- that every culture of P . rhodozyma that he ex-
coccus by sugar fermentation. Weijman et al.67 amined contained a cis-isomer of astaxanthin,
pointed out that given the similarity in cell wall identified by iodine-catalyzed stereomutation, and
composition, Phaffia is distinguished phenotyp- suggested that the cis-isomer represented a gen-
ically from Cryptococcus solely on the basis of uine biosynthetic product of the yeast and not an
fermentation. They questioned the value of gas- artifact of isolation.
eous fermentation as a distinguishing feature since Wild strains of P . rhodozyma so far isolated
some yeasts product ethanol slowly or without contain up to 500 pg/g dry yeast of total caro-
gas. Yamada and Kawasaki6' clarified the phy- tenoid, of which 40 to 95% is astaxanthin. The
logenetic relatedness of Phafia and Cryptococ- content of astaxanthin varies substantially de-
cus by comparison of 18srRNA sequences. Par- pendent on the strain and the method of culture
tial sequences of 18srRNA were determined using (see below). Astaxanthin from P. rhodozyma was
reverse transcriptase for amplification of the iso- found to be deposited in rainbow trout fed prep-
lated nucleic polymers. Significant differences in arations of the y e a ~ t . ' ~ .Yeast
~' cells that were
the fingerprint region of the rRNA nucleotide mechanically disrupted to disrupt the thick cell
sequences were detected between P . rhodozyma wall resulted in good pigmentation when incor-
and Cryptococcus laurentii and Cryptococcus lu- porated into the diets of rainbow trout, but pig-
teolus. Yamada and Kawasaki6*concluded that ment from nondisrupted yeast was not depos-
the genera were taxonomically different. Gueho ite4L7' Astaxanthin was most efficiently deposited
et al.69 analyzed the evolutionary affinities of when yeast was fed that had its cell wall partially
Phafia and other heterobasidiomycetous yeasts removed by enzyme digestion. ' O Although ca-
by 18s and 25s ribosomal RNA sequences and rotenoids were poorly absorbed from whole yeast
concluded that Phafia clustered with Cystojilo- by rainbow trout in the feeding studies of Johnson
basidium capitatum and Trichosporonpullulans. et al. ,70.71 subsequent feeding trials using Atlantic
They hypothesized that P . rhodozyma may derive salmon have indicated that salmon can absorb
from an organism having a primitive dolipore in astaxanthin from whole (nondisrupted) yeast.

304
Pigmentation of lobsters and eggs of laying hens dition to pure isoprenoids, many compounds are
was also observed in laboratory feeding studies derived from mixed biosyntheses including plasto-
using preparations of P. r h o d o ~ y m a . ~ ~ , ~ ~ and ubiquinones, ffavonoids, alkaloids, canna-
binoids , and porphyrins. Furthermore, prenyla-
tion of proteins has been found to be involved in
E. Other Microorganisms mating interactions between fungi and regulation
of mammalian cell growth.*I
Some bacteria, including Mycobacterium Mevalonic acid (MVA) is the first committed
lucticola and a Brevibacterium S P . , and ~ ~ fungi precursor to the 5-carbon isoprene unit that is the
in the genus P e n i ~ p h o r ahave
~ ~ been reported to fundamental building unit for isoprenoid biosyn-
contain astaxanthin. Carotenoid levels are low or thesis (Figure 5). Mevalonate is formed from ace-
growth is slow in these organisms and fermen- tyl CoA and acetoacetyl CoA by hydroxyme-
tation development has not been pursued. The thylglutaric acid (HMG) -CoA synthase and
industrially important xanthophylls, canthaxan- reductase (HMGS and HMGR). Labeled acetate
thin and zeaxanthin, are produced by strains of and mevalonate (the acetate-replacing factor) are
Bre~ibacterium~’ and Flavobacterium,76 but the incorporated into carotenoids in many organisms.
productivity is too low for commercial fermen- Addition of mevalonate to the growth medium
tation. It is likely that other organisms are present dilutes out the incorporation of acetate into p-
in nature that contain astaxanthin and other val- carotene in Phycomyces blukesleanus.82Meva-
uable carotenoids, and samplings of organisms lonate is assimilated into carotenoids about 20
from marine environments and extreme environ- times more efficiently than acetate.83
ments would be useful. Carotenoids function in The mevalonate pathway must be precisely
heterotrophic microorganisms as protectants regulated to provide needed quantities and to avoid
against oxygen radicals and light,77,78and envi- overaccumulation of individual isoprenoids. Ow-
ronments in which radicals may be exacerbated ing to the importance of certain isoprenoids in
such as hot springs or mineral-rich illuminated human health and disease, regulation of the mev-
desert ponds would potentially yield organisms alonate pathway to cholesterol has been studied
with high potential for the production of astax- extensively in higher mammals.80,81 The key reg-
anthin and other valuable xanthophylls. ulatory mechanisms found for cholesterol bio-
synthesis in mammals include feedback regula-
tion by low-density lipoprotein receptors, sterol-
V. ASTAXANTHIN BIOSYNTHESIS AND mediated repression feedback repression of the
ITS REGULATION genes for HMG-CoA synthase and reductase
(HMGS and HMGR), and posttranscriptional
A. Astaxanthin Biosynthetic Pathway - regulation of HMGR. Regulation in mammalian
General Comments cells occurs primarily at steps early in the mev-
alonate pathway that control formation of mev-
Although much elegant work has been done alonate. Mammalian cells have also been shown
on the chemistry of the carotenoids, very little is to govern disposition of mevalonate into various
known concerning carotenoid biosynthesis, par- classes of isoprenoids; when mevalonate is lim-
ticularly the xanthophylls. Astaxanthin is a te- iting in the cell it is preferentially used in high-
traterpenoid produced from the mevalonate path- affinity nonsterol pathways.81Regulation of the
way (Figure 5).79-81Isoprene units containing 5 mevalonate pathway in fungi and plants is poorly
carbons are enzymatically combined into S-car- understood. In the yeast Succhuromyces cerevis-
bon polymers, which can take on many spatial iue and in plants, overall control of the meva-
structures and have many biological functions. lonate pathway also occurs partly by regulation
The direct polymer products include isoprene (C,) of early enzymes, particularly HMGR.84,85 Over-
and its derivatives, monoterpenes (Clo), sesqui- expression of HMGR in S . cerevisiue resulted in
terpenes (C1,), gibberellins (Czo), sterols (C30), striking proliferation of stacked membrane pairs
carotenoids (Ca), and polyrubbers (C,,). In ad- surrounding the yeast nucleus,86indicating that cel-

305
 -4
Geranyl-PP AWP
J4 Iropentenyl-PP

FIGURE 5. The mevalonate pathway to farnesylpyro-

phosphate.*'

lular mechanisms governing the levels of mem- and closely related terpenoids. Astaxanthin and
brane synthesis may be very important in iso- its precursor carotenoids are derived from ger-
prenoid regulation. anylgeranyl pyrophosphate (GGPP) (Figure 6),
MVA formation may partly be controlled in and it would be expected that the formation of
S. cerevisiae by catabolite repres~ion.~' Indirect other terpenoids with GGPP as precursor would
evidence has indicated that HMGR and other early share regulatory controls with carotenoids. Tada9'
steps of isoprenoid synthesis influence carotenoid showed that ubiquinone and carotenoids, which
biosynthesis. In plant systems, the HMGR in- both use GGPP as a precursor, were coordinately
hibitor, mevinolin, decreased carotenoid forma- regulated by light, but that formation of ergos-
tion.88*89 In the yeast Rhodotorula minuta pho- terol, which is biosynthesized via farnesylpyro-
toinduced production of carotenoids was phosphate, was not influenced by light. The reg-
competitively inhibited by mevinolin.gOProvi- ulation of the later steps of the mevalonate
sion of mevalonate to fungi has been demon- pathway to carotenoid biosynthesis in fungi and
strated to increase carotenogenesis.82-83 Several plants is poorly understood at pre~ent.~'.~' Con-
controls affect early steps in the pathway com- trolling factors include compartmentation of en-
mon to all isoprenoids, whereas others only affect zymes and substrates, regulation of oxidative me-
later steps in biosynthesis specific for carotenoids tabolism, repression of regulatory genes by light

306
 4a."-v-.1-v

-
Farnesyl-PP Mop,+

/
,sopent+enyl-pp GeranYlgeranYl-PP Prephytoene-PP

Phytoene Phytofluene

Neurosporene \ C-carotene

Lycopene
-. ',w
.. 8-zeacarotene

w 8-carotene
-*''
Torulene

m
o 3-Hydroxy-
echinenone

Astaxanthin

FIGURE 6. The astaxanthin biosynthetic pathway proposed for Phaffia

rhodozyma . The left vertical pathway (phytoene + trans-astaxanthin) rep-
resents the pathway originally proposed by Andrewes- for strain 67-210.
The right pathway (p-zeacarotene --* torulene + HDCO + DCD + trans-
astaxanthin) represents an alternative pathway based on the detection of
the intermediate carotenoids in strain 67-385 (see text for details).

307
and other environmental factors, kinetic controls after inoculation. GGPP synthase was unstable
of individual biosynthetic enzymes, and devel- and had to be purified very rapidly. The overall
opmental p r ~ c e s s e s . ~ ~ ~ ~ purification was 10,000-fold and the pure en-
zyme had a specific activity of 290 nmol/min/
mg protein. Overall, the protein was unusual for
B. Enzymology of Carotenoid its stringent substrate specificity and low molec-
Biosynthesis ular weight. In Phycomyces, which lacks plastid-
like organelles, farnesyldiphosphate represents
Biosynthesis of carotenoids has been re- the branch point between sterol and carotenoid
viewed r e ~ e n t l y ;only
~ ~ advances
~ ~ ~ - ~ since
~ these biosynthesis. In the fungus, sterol and carotenoid
reviews are discussed below. Carotenoid synthe- formation presumably both occur in the cyto-
sis in fungi is a multistep pathway and various plasm. Therefore, it would be expected that en-
steps are compartmentalized. Synthesis of MVA zyme affinity would be a key for the regulation
and its conversion to GGPP is probably mediated of the two pathways in fungi.
by soluble enzymes in yeasts and fungi, although GGPP undergoes tail-to-tail condensation to
membranous structures were shown to form in form the first C,, carotene, phytoene
endoplasmic reticulum in S. cerevisiae mutants (7,8,11,12,7' ,8', 11' ,12'-octadehydro-V,'P-car-
that overexpressed HMGR, 86 Carotenogenic en- otene), via the intermediate prephytoene-PP (Fig-
zymes in fungi (phytoene synthase and succes- ure 6). 15-cis-Phytoene is the most common geo-
sive enzymes) are membrane bound and have metric isomer detected.95 Recently the enzymes
been found in microsomes and possibly in mi- catalyzing the synthesis of phytoene from iso-
tochondria. Extensive compartmentalization gives pentenyldiphosphate have been isolated and char-
much complexity to carotenoid biosynthesis and acterized in plant^.^^.'^ Prephytoene and phy-
regulation and it is possible that isoprenoid syn- toene are formed by a monomeric protein (47,500
thesis occurs coordinately in several cellular Da) in Capsicum chromoplasts'OOthat has bi-
locations. functional enzyme activities, i.e., the coupling
Carotene biosynthesis in cell-free extract^^^-^^ of two GGPP molecules to prephytoene phos-
has been demonstrated in various plants; in fungi, phate and conversion of prephytoene phosphate
including Phycomyces blakesleanus, Neurospora to phytoene. The specific activity of the purified
crassa, and Blakeslea trispora; and in various enzyme was 4000 nmol of geranylgeranylpyro-
bacteria, particularly in Myxococcus and photo- phosphate incorporated into phytoene per milli-
synthetic bacteria (Rhodobacter and Aphano- gram protein in 1 h. The enzyme was strictly
cupsa). Geranylgeranylpyrophosphate (GGPP) dependent on MnZ+ , and no other divalent cation
synthase has been purified from chromoplasts of stimulated activity. The preference of the enzyme
and, more recently, from the caro- for Mn2+ over Mgz+ may contribute to the pref-
tenogenic fungus Phycomyces blakesleanus.97 The erential conversion of GGPP to carotenoids than
enzyme from Phycomyces was reported to be a other terpenoids. Inorganic pyrophosphate was
dimer consisting of two 30,000 M, subunits. The also a strong inhibitor in vitro of phytoene bio-
homogenous enzyme had Michaelis constants of synthesis. The finding by Dogbo et al.Im of a
9 p M for isopentenyldiphosphate and 60 pii4 for bifunctional enzyme may be a precedent for the
farnesyldiphosphate. The enzyme is distinct from discovery of enzymes catalyzing multiple cata-
a farnesyldiphosphate synthase previously iso- lytic activities in carotenoid biosynthesis.
lated from S. cerevisiae and from l i ~ e r .Strains
~~.~ In many organisms, phytoene is desaturated
of P. blakesleanus differing in carotenoid accu- by a series of four didehydrogenations to lyco-
mulation also had different activities of the syn- pene (Figure 6). Neurosporene is an intermediate
thase. A P-carotene-overproducingstrain of P. in this series of desaturations. Phytoene desatu-
blakesleanus produced four times the level of rase catalyzes the formation of two double bonds
GGPP synthase of the wild-type strain.97 The at C , , and C1,,,producing the first C, carotene.
maximum rates of in vitro GGPP synthase activ- Feedback regulation of the enzyme has been dem-
ity and carotene production occurred 24 to 40 h onstrated, and in certain fungi formation of the

308
enzyme activity is also induced by light. Phy- conidiation and during germination of the coni-
toene dehydrogenase may be an important en- dia, indicating that differentiation is involved in
zyme for control of the pace of carotenoid syn- regulation of carotenoid biosynthesis in N . crassu.
thesis. Recently, phytoene desaturase was Beyer et a1.'09 proposed that molecular ox-
detected immunologically in algae and higher ygen is essential for carotene desaturation and
plants with a polyclonal antiserum raised against cyclization in daffodil chloroplasts. They pro-
a phytoene-desaturase-P-galactosidasefusion posed that 0, acted as an electron acceptor and
protein produced in Escherichia coli. lo' The fu- that an oxidoreductase served as a redox mediator
sion protein was prepared as a 921 bp segment between phytoene desaturase and 0,. These re-
of the crtl gene in the carotenoid gene cluster sults could help to explain the physiological ob-
from Rhodobacter capsulatus. 102-105 Attempts to servations that carotene synthesis is decreased in
purify phytoene desaturase have been very dif- many organisms when oxygen and light are
ficult due to lability of the enzyme, and little is limiting.
known of its structural and regulatory properties. Although in vitro studies have indicated that
Isolation and genetic characterization of mutants lycopene and neurosporene cyclize to P-carotene
in fungi and in bacteria have indicated that one and p-zeacarotene, respectively, the cyclase en-
enzyme is sufficient for the dehydrogenations in zymes have not been isolated. The enzymes are
some organisms, whereas two enzymes are needed membrane bound and difficult to purify from the
in ~ t h e r s . ~In~Neurospora,
,'~ one enzyme is suf- plastids of plants .92 Lycopene cyclase activity
ficient, whereas in R. capsulatus genetic evi- was demonstrated in chromoplast membranes of
dence suggests that two enzymes are needed. The Capsicum but the enzyme has not yet been pur-
genes and gene products mediating zeaxanthin ified. l lo Attempts to isolate mutants of plants and
biosynthesis in Erwiniu uredovora were deter- microorganisms that accumulate lycopene or
mined by cloning and functional expression in neurosporene have been relatively unsuccess-
Escherichia coli. '08 Only one gene product (CrtI) f ~ l . In
~ , Phycomyces the cyclase gene is asso-
was required for conversion of phytoene to ly- ciated with another function in the curRA bi-
copene. Recently, the uZ-1 gene encoding phy- functional gene.'" Hence, it is possible that
toene dehydrogenase of Neurospora crassu was cyclization may occur on an enzyme complex in
cloned and its structure and regulation ana- fungi. A single gene product (CrtY) was iden-
lyzed. l" The gene was closely linked to homo- tified from Erwiniu uredovora that cyclized ly-
serine dehydrogenase, which provided a select- copene to p-carotene. Io8
able marker for its expression. Sequencing the Very little is known about the genes and en-
al-1 gene and analysis of genomic and cDNA zymes that mediate the biosynthesis of xantho-
nucleotide sequences indicated an open reading phylls from carotene precursor^.^^ Hydroxylation
frame of 1788 nucleotides encoding a 595-resi- occurs late in carotenogenesis and involves mixed
due polypeptide with predicted molecular weight function oxidase (MFO) reactions. l I 2 In vitro bio-
of 66 kDa. The C-terminal region has a hydro- synthesis of P-cryptoxanthin from p-carotene in
phobic region, indicating that it could be mem- Aphanocupsa membranes suggested involvement
brane associated. Two introns were identified by of a monooxygenase reaction. Hydroxylation
comparison of the genomic and cDNA se- was dependent on 0, and sensitive to KCN and
quences. Extensive homology was found be- monooxygenase inhibitors. Astaxanthin forma-
tween al-1 and crtD that encode phytoene de- tion in P . rhodozyma is inhibited by metyrapone
hydrogenase and neurosporene dehydrogenase of and piperonyl butoxide. 118*'68 These two com-
R. cupsulutus. Comparison of the three desatu- pounds inhibit mixed function oxidase reactions
rases indicated homology and a conserved nu- involving cytochrome P450s. A single gene prod-
cleotide-binding fold common to other flavopro- uct (CrtZ) was postulated to convert @-carotene
tein dehydrogenases. lo' Studies of photoregulation to zeaxanthin in Erwinia uredovoru,'08 but the
indicated that al-I mRNA increased in response oxygenase has not been isolated and character-
to blue light.'" The authors also reported prelim- ized. Much work needs to be done on the bio-
inary data that al-1 mRNA accumulated during synthesis of xanthophylls in fungi.

309
C. Astaxanthin Biosynthetic Pathways of rulene and DCD have been confirmed by mass
Haematococcus pluvialis and Phaffia spectroscopic analysis of the purified pig-

-
rhodozyma ments. 1 1 7 * 1 1 Isolation
8 of these carotenoids par-
tially illustrated the biosynthetic pathway to
The biosynthetic pathways from phytoene to HDC0:torulene 4-ketotorulene 3 etc. (Figure
astaxanthin have been indirectly inferred by the 6). The detection of /3-carotene and torulene in
chemical identification of various carotenoids in yellow (p-carotene-accumulating) mutants of P.
the organisms. Generally, pathways are proposed rhodozymu117*118 indicated that these carotenes
based on a logical sequence of biosynthetic events: might be the starting components of two xantho-
desaturation --* cyclization 3 oxygenation. The phyll (astaxanthin) biosynthesis pathways, one
enzymes catalyzing these steps have not been of which has a bicyclic precursor (as recognized
isolated in astaxanthin-producing organisms and by Andrewes) and the other a monocyclic pre-
the actual sequence of reactions and precursors cursor. Studies with inhibitors of carotenogenesis
is not known with certainty. The patterns of as- have also supported the presence of two biosyn-
taxanthin biosynthesis in Haemutococcus and thetic routes to astaxanthin"* Yeast cultures in-
Phafia appear to be different. In H . pluvialis, cubated with 2-methylimidazole (MI) or triethy-
Goodwin and J a m i k ~ r nidentified
~~ p-carotene lamine hydrochloride (TEA) accumulated HDCO
and lutein in the green phase of the organism. and had reduced quantities of astaxanthin, indi-
As nitrogen became limiting, the autotrophic cells cating that astaxanthin may be produced from
transformed to the red and brown and red phases HDCO. Further research is needed to elucidate
during which astaxanthin accumulated. In a later the complex pathway of astaxanthin formation in
study Donkin1I4studied carotenogenesis in more P . rhodozymu.
detail in H . lacustris and found that the alga
utilized acetate-[2-I4C]and produced p-carotene,
probably via the intermediates echinenone and
canthaxanthin. The yeast P. rhodozyma appears D. Chemical Inhibition of Carotenoid
to synthesize astaxanthin from p-carotene using Biosynthesis
different intermediates. Andrewes et al .45 pos-
tulated that P-carotene is first converted to echi- Studies with inhibitors of the isoprenoid
nenone, and echinenone is then hydroxylated to pathway have been very useful in understanding
3-hydroxyechinenone (3-hydroxy-p,p-caroten-4- the biosynthesis of carotenoids and for isolation
one). This intermediate is oxidized to phoeni- of hyperproducing strains, The principal inhibi-
coxanthin (3-hydroxy-P,@-caroten 4,4'-dione), tors used affect various steps of carotenoid syn-
which is further hydroxylated at C-3' to give thesis:l19-122
astaxanthin (Figure 6). Other possible interme-
diates in the biosynthesis of astaxanthin, includ- 1. Phytoene desaturase (phenylpyridazinones,
ing isocryptoxanthin, canthaxanthin, and 3,4'- e .g ., norflurazon, diphenylamine, p-ionine)
dihydroxy-P,P-carotene-4-one, were not found 2. (-Carotene desaturase (e.g., amitrole, N -
in P . rhodozyma. However, Andrewes et al.45 methyl-carbarnate, methoxyphenone)
also detected the carotenoid HDCO, and could 3. Lycopene cyclase (substituted triethylam-
not explain its biosynthesis in the proposed path- ines, e.g., CPTA, a-picoline, nicotine,
way. In 1979, Johnson and Lewis115detected p- imidazoles)
zeacarotene in P. rhodozyma under stressful low 4. p-Carotene hydroxylase (MFO inhibitors,
pH conditions, and they proposed a bypathway e .g ., piperonylbutoxide and metyrapone)
from neurosporene to p-carotene through y-car-
otene. An et found two previously unde- Many of the inhibitors cause striking visual
tected carotenoids in P. rhodozyma that were ten- changes in the organisms, such as bright red pig-
tatively identified as 3,3' -dihydroxy-p ,y- mentation when lycopene accumulates in the
carotene-4,4' -dione (DCD) and torulene,117.1 I s presence of cyclase inhibitors. Ninet et a1.'23re-
that accumulate carotenes. The structures of to- ported that MI inhibited synthesis of @-carotene

310
from lycopene and slightly increased total caro- cellularly in organelles that may generate radi-
tenoid in Blukesleu trisporu. Elahi et al.Iz4 also cals, for example, chromoplasts or mitochondria,
reported that MI inhibited p-carotene accumu- or in the surface membranes of organisms ex-
lation but stimulation accumulation of lycopene posed to high intensities of light. Carotenoids are
and phytoene in Phycomyces blakesleunus. In- often membrane associated, as indicated by de-
creased carotenoid concentrations observed in tailed biochemical analyses of membranous
Phycomyces in the presence of inhibitors sup- structures in cyanobacteria. There are probably
ported the hypothesis of Cerda-Olmeda and co- three distinct membranous systems in cyanobac-
w o r k e r ~ that
' ~ ~ accumulation of p-carotene (the teria (photosynthetic thykaloids, and the inner
end product) caused feedback inhibition of car- and outer cytoplasmic membranes). 132 Mitochon-
otenoid biosynthesis in this fungus. dria in Phycomyces spores have been reported to
TEA inhibited synthesis of p-carotene from contain c a r ~ t e n o i d s 'and
~ ~ detailed studies have
acyclic carotenoids in B. trisporu. lZ6 Hsu et al. indicated that the mitochondria1 outer membrane
also reported that CPTA (a derivative of TEA) of Neurosporu crussu contains carotenoids.134 In
slightly reduced p-carotene but increased lyco- some organisms, carotenoids are located in lipid
pene up to 150-fold and total carotenoid up to 3- globules and not in mitochondria. 135-137 Consid-
fold. Addition of cycloheximide (an eukaryotic erable amounts of carotenoids are located in an
protein synthesis inhibitor) together with CPTA endoplasmic reticulum fraction from N. crussu. 13'
prevented lycopene accumulation. Benedict et Many classes of lipids, including certain steroids
al. IZ7 also found increased carotenoid formation and other terpenoids, are synthesized in the
when 2-(4methylphenoxy) triethylamine (MPTA, smooth endoplasmic reticulum in various organ-
related to CPTA) was used as an inhibitor. The isms. 139 Following export from the endoplasmic
findings indicated that CPTA or MPTA might reticulum, carotenoid synthesis may take place
derepress the synthesis of carotenogenic enzymes. in the membranes or on the surface of these
In our laboratory, yeast cultures of P . rho- globules.
dozymu incubated with MI or TEA did not ac- P . rhodozymu cells were analyzed in our lab-
cumulate lycopene but HDCO and produced nor- oratory for carotenoid location by laser confocal
mal amounts of p-carotene. 118 These data suggest fluorescence microscopy (LCFM). The fluo-
that P . rhodozymu has two types of cyclases. We rescence images obtained by LCFM of wild-type
have also observed that mutants increased in car- cells and a carotenoid hyperproducer were strik-
otenoid synthesis are changed in resistance to MI ingly different (Figure 7). Autofluorescence de-
and TEA and have selected carotenoid hyper- tected in P . rhodozymu cells by LCFM was not
producing mutants on YM agar supplemented with caused by flavin or nucleotide cofactors essential
the inhibitors. for growth since white mutants fluoresced very
poorly in the LCFM andysis. Also, the excitation
and emission wavelengths used for the analysis
of carotenoids by LCFM do not readily detect
E. Cellular Location of Carotenoids flavins and nucleotides. The carotenoids in P .
rhodozymu were also not located in mitochondria
The cellular location of carotenoids is of con- or endoplasmic reticulum, which was demon-
siderable importance for the development of mu- strated by staining with DiOC6 (3,3'-dihexylo-
tants that produce high quantities of carotenoids. carbocyanine iodide), 14' followed by photo-
Carotenoids are intracellular products and the bleaching and comparison of fluorescence patterns
maximum concentration will depend on cellular before and after bleaching. Autofluorescence and
location, for example, if they are confined to DiOC, were observed as green and red areas in
organelles or are distributed throughout the cell the yeast cells. Autofluorescence and fluores-
in lipid globules. Considerable evidence indi- cence of DiOC, did not overlap. These results
cates that carotenoids protect cells against pho- indicated that carotenoid was not located in mi-
tosensitizers and oxygen radicals. lZ9-l3' It would tochondria or endoplasmic reticulum. Carotenoid
be expected that carotenoids are located intra- fluorescence images obtained by LCFM were

311
 compared to subcellular structure analyzed by
transmission electron microscopy (TEM), 169 Lipid
globules and membranes were stained with 0s-
mium tetroxide, lead citrate, and uranyl acetate.
The stained lipid globules were matched to the
cellular location of fluorescent bodies observed
by LCFM. Older cultures of P . rhodozyma con-
tained more carotenoid than younger cultures and
large numbers of lipid globules that were asso-
ciated with the cytoplasmic membrane. Young
cells contained fewer numbers of relatively large
lipid globules. The large lipid globules in caro-
tenoid hyperproducers appeared to fragment and
migrated to the membrane at an earlier stage of
growth than the wild-type.
Images from TEM analysis of P . rhodozyma
indicated that older cells and carotenoid hyper-
producing mutants (CHMs) contained mitochon-
dria of different morphology than younger cells
or strains that produced low levels of carotenoid.
Hyperproducing strains appeared to contain
smaller and larger numbers of mitochondria than
A
the wild-type. These results suggest that an early
step of carotenogenesis in P . rhodozyma may be
associated with mitochondria and that lipid glob-
ules contain carotenoids that become dispersed
to the cell membrane as the cells age.
Analysis of P . rhodozym strains by LCFM'"O
has provided insight into the cellular location of
the pigments and also has enabled a semiquan-
titative calculation of the maximum quantity of
intracellular carotenoid that could be attained in
P . rhodozyma. The average fluorescence ob-
tained by LCFM is related to the carotenoid con-
tents of cultures. Carotenoid fluorescence varies
considerably depending on the yeast strains and
age. The semiquantitative value of maximum car-
otenoid content was calculated based on analysis
of cells in culture that contained high fluores-
cence. Fluorescence per unit area of yeast (1-pni
sections) was found to be proportional to caro-
tenoid per gram of yeast. Calculation of maxi-
mum carotenoid as a function of fluorescence
gave a maximum carotenoid yield of 15 mg/g
yeast based on extrapolation of a plot of caro-
tenoid as a function of fluorescence.'70This is a
B maximum concentration similar to that obtained
for p-carotene in mutants of Phycomyces.'*'
FIGURE 7. LCFM images ofPhaffia rhodozyma yeast
cells grown for 10 d in YM medium at 20°C. (A) Strain
The primary carotenoids in eukaryotic algae
67-385(wild-type). . . Strain 2A-34. See Reference
_ . . (B) involved in photosynthesis are located in the thy-
140 and text for details. kaloids, where they are associated with chloro-

312
phyll. 142 Carotenoids that accumulate during en- otenogenesis in the yeast. In R . rubru carotenoid
cystment may be located in the cytoplasm. synthesis occurs in three main phases? (1) a
Astaxanthin in Huemutococcus spp. accumulates period of active synthesis leading to maximal
in the cytoplasm. 143 Interestingly, astaxanthin ap- concentration, (2) a period of persistence during
pears to initially accumulate in the vicinity of the which the concentration stays relatively constant,
n u c l e u ~ , 'possibly
~ ~ ~ ' ~in~ endoplasmic reticulum, and (3) a period during which the pigments grad-
where S . cerevisiue mutants that overproduce ually disappear from the yeast. This pattern is
HMG-CoA reductase have been shown to possess also found in leaves of trees, the onset of caro-
large quantities of stacked membranes in endo- tenoid formation becoming rapid with the ap-
plasmic reticulum surrounding the nucleus. pearance of the first leaves, a climax, and then
a gradual senescence in autumn. GoodwinS2
pointed out that in fungi grown in surface cultures
F. Environmental Regulation of pigment synthesis does not begin until growth
Astaxanthin Biosynthesis has stopped, and carotenoid formation is stimu-
lated by excess carbohydrate and limiting
Astaxanthin production is regulated by sev- nitrogen.
eral environmental factors, including light, aer-
ation, nutrition, and other factors in Huemuto-
coccus and Phafiu. Many of these effects are 2. Light
not specific and the change is due to alterations
of overall cellular metabolism. The influence of Light is important for the regulation of car-
environment on carotenoid formation, particu- otenogenesis in a wide variety of organisms. In
larly astaxanthin, is reviewed in this section. several fungi light together with oxygen acts as
a specific inducer of carotenogenesis. Growth
and pigmentation of P . rhodozymu was inhibited
1. Kinetics of Growth and Pigment by high light intensities. 146 Carotenogenesis in P .
Formation rhodozymu was induced by lower light intensi-
ties. Blue light induced higher pigmentation than
Astaxanthin content varies significantly dur- red, yellow, or green light when the yeast was
ing the life cycle of Huemutococcus.'45 Astax- grown on the surface of YM agar at 7.5"Cfor 1
anthin is not found in growing vegetative cells, 145 month.'& The light response of the yeast was
but is formed during encystment of the alga (see increased when it was exposed to antimycin, a
below). Astaxanthin is formed during growth of respiratory chain inhibitor that may increase the
P . r h o d o z y m ~ , " ~ -but
" ~ continues to be synthe- formation of oxygen radicals. Carotenoid for-
sized after growth has stopped. The availability mation and growth was especially increased in
of carbon source during this nongrowth phase is antimycin + light.146Experiments in our iabo-
important for astaxanthin synthesis since cells ratory have indicated that carotenoids may be
suspended in medium or buffer without carbon formed in response to intracellular oxygen radical
do not increase in astaxanthin content, but do in formation. 1 1 6 ~ 1 4The
7 addition to yeast cultures of
media containing carbon or spent media from a duroquinone (DQ) , a redox-cycling quinone
fermentation (unpublished results). It is likely known to generate intracellular superoxide, sub-
that P . rhodozymu excretes a carbon intermediate stantially increased astaxanthin formation. 147 In
during growth, for example, an alcohol, acetic an earlier study, carotenoids were demonstrated
acid, or a citric acid cycle intermediate, which to protect the red yeast Rhodotorulu mucilugi-
is later reassimilated and stimulates nosu against oxygen radicals generated by dur-
carotenogenesis. oquinone.148R. muciluginosa was found to con-
Carotenoid biosynthesis in the yeast Rho- tain only one superoxide dismutase (SOD), the
dotorulu rubru was shown to occur mainly after Mn type. In contrast, Succharomyces cerevisiue
growth had ~ t o p p e d , ' ~suggesting
,~~ that nutrient contain Fe and CdZn SODS. The lesser capacity
depletion or changes in physiology triggered car- of R. rnuciluginosa to detoxify superoxide and

313
its derivative radicals could be related to for- not been demonstrated to substantially affect as-
mation of carotenoids as protectants. Carotenes taxanthin formation in P . rhuduzyma, although
are well known to react with radicals, and re- mutants of P . rhuduzyrna with decreased ability
cently T e r a ~ 'showed
~ that oxygenated carot- to assimilate nitrogen were increased in carot-
enoids, particularly canthaxanthin and astaxan- enoids. Limitation of nitrogen in the presence
thin, also react efficiently with oxygen radicals. of ample glucose could increase the proportion
Hence, the primary function of astaxanthin and of carbon incorporated in lipid. Control of the
related carotenoids in P. rhuduzyma and other availability of carbon is critical for astaxanthin
organisms may be to protect against oxidative synthesis in P. rhoduzyma. In most fermenta-
damage, and synthesis may be induced by oxygen tions, it is important to slowly feed sugar glucose
radical stress. to limit fermentative metabolism and alcohol pro-
Light is also known to stimulate astaxanthin duction and to stimulate oxidative metabolism.
formation in Haematucuccus pluvialis. 1 1 4 * 1 4 9 Leucine and valine can stimulate carotenogenesis
calculated that astaxanthin synthesis was in certain fungi by means of their conversion to
seven times higher in light. Goodwin114showed 3-hydroxy-3-methylglutaryl-CoA,but these
that H . ptuvialis cultures placed in the dark de- amino acids to do not seem to be carotenogenic
creased in carotenoid content and did not change in P . rhoduzyma.
from the green to red or brown phases.

5. Other Nutrients
3. Temperature
Fluoride stimulates carotenogenesis in cer-
P . rhoduzyma grows in the mild temperature tain fungi ,73 presumably by inhibiting phospha-
range of 0 to 27°C.63Johnson and Lewis"' found tases that hydrolyze phosphorylated intermedi-
that yeast yields decreased above 22.5"C, the ates in the isoprenoid pathway. In light of recent
optimum for growth The carotenoid con- findings that phosphorylation of key regulatory
tent of the yeast was fairly constant from 14 to proteins controls expression of the isoprenoid
26OC.Il6The inability of the yeast to grow above pathway,80 it is possible that altering the phos-
27°C is a serious drawback to its industrial de- phoryl transfer to key proteins could also con-
velopment. Several research groups have not been tribute to the fluoride effect. Phosphate has not
successful in isolating PhufJia mutants that grow been reported to influence carotenogenesis in P.
at temperatures >30°C. rhodozyma.
Certain psychotropic strains of Rhodotorula Limitation of certain micronutrients, includ-
accumulate carotenes at low temperatures but ing manganese, iron, and sulfate, enhances sec-
synthesize torulene and tomlarhodin as the tem- ondary carotenoid formation in some microal-
perature is raised. Temperature has been re- gae.'49,*51Manganese and iron were demonstrated
ported to have little impact on carotenoid for- to affect carotenoid formation in P . rhuduzyma
mation in green algae.149 when it was grown on s ~ c c i n a t e , possibly
'~~ by
exacerbation of oxygen radical formation. Other
factors that may affect oxygen radical formation
4. Nitrogen and Carbon and destruction would be expected to affect car-
otenogenesis. To our knowledge, systematic
In the Chluruphyceae, nitrogen limitation is studies of the effects of micronutrients have not
a key factor for accumulation of a~taxanthin.~~ been carried out on P . rhodozyrna.
Acetate and glycine were demonstrated to stim-
ulate astaxanthin formation in H . pluvialis,
whereas other nitrogen and carbon sources de- VI. STRAIN IMPROVEMENT
layed the onset of p r ~ d u c t i o n . Hence,
~ ~ ' ~ the
quality and availability of carbon and nitrogen A. Genetic Approaches and Limitations
are critical for carotenogenesis in H . pluviulis.
To our knowledge, nitrogen limitation has Currently, industrial astaxanthin production

314
by P . rhodozyma is limited by a lengthy low- enoids. I l 7 Spheroplasts can be readily obtained
temperature fermentation and especially by the in PhafJia with Trichoderma lytic enzymes, and
unavailability of stable astaxanthin high-produc- it may be possible to develop strains by Sphero-
ing strains. The best available natural strains pro- plast fusion with increased carotenoid. More work
duce only -500 p.g total carotenoid per gram of needs to be done in this area using selective mark-
yeast, and strain development is required to in- ers. To the authors’ knowledge, gene cloning for
crease the astaxanthin content and possibly the increasing astaxanthin production has not been
maximum temperature for growth. In contrast, attempted. Cloning and overexpression of the
microalgae are often prolific natural producers of genes encoding astaxanthin formation would be
carotenoid and several strains of Haemutococcus difficult since several genes are involved in bio-
and related green algae naturally produce from synthesis and genetic regulation is unknown. This
2000 to 20,000 pg carotenoids per gram cell and approach would potentially be fruitful if rate-
strain development for industrial production of limiting enzymes and precursors were identified
carotenoid is not as critical. in the astaxanthin pathway and specific gene
Three approaches can be used for genetic products were highly expressed. Analysis of car-
improvement of industrial asexual yeasts such as otenogenic enzymes in vitro in highly caroten-
P . rhodozyma: mutagenesis, recombination of ogenic mutants of other fungi have shown that
mutants (e.g., by protoplast fusion), and gene activities of specific enzymes were increased,
cloning and amplification. Each approach has PO- e.g., phytoene desaturase.152
tential benefits and limitations. Mutagenesis is
often used because of simplicity, and it is not
necessary to have considerable knowledge of the 6. Screening
biosynthesis and genetic regulation of the desired
product. The success of this method depends on 1. General Comments
the efficiency of mutagenesis and proper screen-
ing. Fortunately, the color of astaxanthin is Screening of pigmented yeast colonies by
orangehed and yeast mutants can be screened by visual examination has limited utility and is often
visual examination. Our laboratory has used vis- not capable of detecting CHMs, since the mu-
ual screening after mutagenesis with UV light, tation rate is low and the difference in carotenoid
ethyl methanesulfonate (EMS), N-methyl-N’-ni- contents between CHMs and parent is usually
tro-N-nitrosoguanidine (NTG), ethidium bro- small. As a series of CHMs is generated in a
mide, and a ~ r i f l a v i n . ”EMS
~ * ~ ~and
~ NTG were laboratory, it becomes increasingly difficult to
the most effective, but after several consecutive obtain higher-producing, stable strains. More
uses of these mutagens, isolation of CHMs be- sensitive methods than visual examination or
came very difficult and isolated CHMs were usu- conditions that selectively detect CHMs are
ally unstable. Without genetic selections and fur- required.
ther knowledge of carotenoid function in the yeast, Astaxanthin is produced as a secondary me-
the problem of instability will be very difficult tabolite in P . rhodozyma and growth of white or
to solve. We have found that CHMs have dif- yellow mutants were not different from that of
ferent physiological properties and are more re- the red parent. 117,118 Growth was also unaffected
sistant to certain chemical agents that have helped in P . rhodozyma treated with piperonylbutoxide,
in the development of genetic selection of CHMs which inhibits carotenoid formation, giving very
(see next section). pale cells.118Since carotenoid production is non-
Recombination by protoplast fusion can be essential for growth under most conditions, and
used with asexual yeasts if more than two dif- CHMs grow more slowly than their parents, ge-
ferent lines of strains are available. Several dif- netic selection is usually not possible for hyper-
ferent natural isolates of P . rhodozyma contain production and development of screening meth-
different quantities and compositions of carot- ods is required.

315
2. Negative Screening formation. IS3 After a null mutation was obtained,
it was possible to isolate S. cerevisiae mutants
CHMs of P . rhodozyma were more suscep- that greatly overproduced HMG-CoA reductase.
tible to certain environmental stresses than wild- It is possible that inhibitors of HMG-CoA re-
type cells. I l h * l l 7 Interestingly ,antimycin-induced
ductase could also be used to isolate CHMs in
mutants were more susceptible to antimycin than P . rhodozyma, but attempts in our laboratory have
parents, even though they were isolated on agar not been successful.
plates containing antimycin. CHMs isolated by
EMS or NTG but without exposure to antimycin
were also more susceptible than their parents to 6.Positive Screening
H,O,' l6 and respiratory inhibitors (antimycin,
KCN, and thenoyltrifluoroacetone). l6 Surpris- Positive screening can be used when an in-
ingly, CHMs were more susceptible than parents hibitor of metabolite synthesis also inhibits growth
to UV light and to photosensitization,'16+1'7-'46 and colonies resistant to the inhibitor are in-
even though carotenoids are believed to protect creased in the content of the desired metabolite.
against light in many organisms. 12y-131 Increased This method requires much less labor than neg-
sensitivity to environmental factors (negative ative screening. An1'' found that MI and TEA
screening) can potentially be used for isolation inhibited cyclization of HDCO in P . rhodozyma.
of CHMs by replica plating or other methods after Interestingly, CHMs were more resistant to TEA
genetic changes. However, negative screening or MI than the parent. 1' These two carotenogenic
after mutagenesis is not recommended because inhibitors were used for positive screening of
the mutational rate to CHMs was found to be CHMs, and strains have been isolated that pro-
very low (<0.006%).'17.'40 duce -3000 pg carotenoid per gram of yeast.
Recently, Lewis et al.Is4 used p-ionone to
isolate to select astaxanthin-overproducing mu-
3. Positive Screening with Inhibitors of tants of P . rhodozyma strain UCD-FST-67-210.
Carotenogenesis p-Ionone was found to inhibit xanthophyll for-
mation, and a colony isolated from a p-ionone-
As discussed above, many compounds in- containing plate (lop4M) and grown in shake
hibit carotenogenesis in various organisms. In- flasks for 5 d produced 725 pg astaxanthin per
hibitors have been used successfully in our lab- gram of yeast (the parent [67-2101 produced 280
oratory to isolate mutants of P . rhodozyma Wg).
affected in carotenogenesis.

4. Flow Cytometry and Cell Sorting

a. Inhibitors of Early Steps in the
lsoprenoid Pathway Our laboratory has successfully used flow
cytometry/cell sorting (FCCS) to isolate
Formation of MVA from HMG catalyzed by astaxanthin hyperproducing mutants of P . rho-
HMG-CoA reductase is the first reaction unique dozyma. Flow cytometry was first developed
to isoprenoid biosynthesis, and is a key rate-lim- in 1934 to count microscopic cells, and Coulter
iting step in isoprenoid biosynthesis in plants, developed an instrument, the Coulter Counter@,
fungi, and mammals. The fungal metabolites, that counted and analyzed cell size. IS6 Fulwyleris7
compactin and mevinolin and related derivatives, further modified the Coulter Counter@in order
are potent inhibitors of this reaction. Mevinolin that it could sort single cells. Contemporary flow
has a Ki value of 3.5 nM for the enzyme from cytometers simultaneously analyze cells for for-
Saccharomyces cerevisiae; however, S . cerevis- ward scatter (related to cell size), side scatter
iae has two alleleic genes (HMGR1 and HMGR2) (related to cell granularity), and for fluorescence
and it is first necessary to obtain a null mutation emissions at three to four different wavelengths.
in one of the genes to substantially affect sterol Modem instruments analyze and sort cells at the

316
speed of 4000 to 10,000 cells per second (the C. History of Strain Improvement of P.
limiting factor is computer speed for analysis and rhodozyma in Our Laboratory
decision making).
Flow cytometry has been used in several areas Our laboratory has used mutagenesis and
of biological research, particularly in clinical ap- screening to isolate a number of mutants affected
plications.'59It has also been suggested that FCCS in carotenoid synthesis (Figure 10). We fust chose
be used for the recovery of rare subpopulations the highest carotenoid-producing strain, UCD-
of organisms that overproduce specific metabo- FST-67-385, from available natural isolates.
lites valuable in industrial microbiology.159-16'To Next, antirnycin induction'I6 was carried out,
be used for practical screening in strain devel- giving stable CHMs up to threefold increased in
opment, the flow cytometer must be able to detect carotenoid. The third step was NTG mutagene-
specific wavelengths of fluorescence to avoid sis."6 Two consecutive NTG mutageneses and
quenching by autofluorescent metabolites in the screening by visual examination produced strains
cells. Attempts to isolate hyperproducing mu- AAN-4 and 2A2N from 2A-2AP. We further mu-
tants by FCCS have often been unsuccessful due tated strain 2A2N with NTG and isolated ANF-
to low fluorescence by the desired compounds 1P and 2F-1 by FCCS. These strains, however,
and quenching by flavin and pyridine nucleotide were unstable and produced less carotenoid than
components. the parent strain 2A2N. Further work needs to
An et al. 140 recently reported successful en- be done to determine the basis for instability in
richment of rare CHMs of P . rhodozymu by FCCS many of the astaxanthin-hyperproducing strains.
after mutagenesis. Yeast carotenoids autoflu- It would also be useful to screen new P . rho-
orescenced weakly when they were activated by dozyma strains isolated from nature for caroten-
light at the wavelengths of absorbance maxima oid content and to develop new strain lines that
(Figure 8). Autofluorescence of P . rhodozyma could be recombined with available mutants. A
carotenoids was low but experimental conditions summary of strains developed in our laboratory
were developed to detect single cells with en- up to December 1990 is presented in Figure 10.
hanced carotenoid autofluorescence. In a simu-
lation test with mixtures of wild-type yeast and
CHM, conditions were devised using FCCS that .
VII INDUSTRIAL ASTAXANTHIN
gave highly enriched populations of CHMs . Mu- PRODUCTION
tated populations exhibited considerable varia-
tion in forward scatter and autofluorescence (Fig- In this section, current production methods
ure 9). Sorting of cells possessing higher for astaxanthin from Huematococcus and PhafJia
fluorescence (in windows I and 11) resulted in rhodozyrna are described briefly.
substantial enrichment of CHMs; 0.6% of sorted
cells in window I and 56% of sorted cells in
window I1 were CHMs, which were detected by A. Haematococcus
plating for single colonies and assay of carotenoid
in the isolated clones. Visual screening and anal- Astaxanthin accumulates to high levels in
ysis of individual colonies from agar plates Huemutococcus and other microalgae in the
showed that only 0.006% of NTG-treated cells Chlorophyceae (0.2 to 2% of dry weight of the
were CHMs. Therefore, FCCS enabled up to algae). An alga undergoing considerable indus-
10,000-fold enrichment of CHMs. FCCS also trial development is the sweetwater organism,
has been used successfully for the enrichment of Huematococcuspluvialis. It is available from the
P-carotene-producing algae populations from a Scripps Institute of Oceanography, La Jolla, CA.
mixture of carotenoid-containing and carotenoid- The encysted cells are used for maintenance
negative populations. 16* Future developments in of the strain. An axenic starter culture is prepared
FCCS , including optimization of sorting win- in a defined medium, often Bold's basal medium
dows and single cell deposition, could further supplemented with thiamin, urea, and sodium
improve the method. acetate. The algae are grown to a density of about

317
 I. *.-
I

I'
400
I
435
I
470
I

505
I

540
I
575
J
610

160 585 510 535 560 585 I0

Wavelength (nm)
FIGURE 8. Visible absorption spectrum of P. rhodozyma (strain 67-385)
and fluorescence emission spectra of purified carotenoids and a carotenoid
extract of P. rhodozyma. See Reference 140 and text for details.

1 to 5 x lo5cells per milliliter under either auto- tial for contamination, cultivation is usually car-
or heterotrophic conditions. The vegetative cul- ried out by a batch process. After the pond is
tures appear as green, motile, flagellated single cleaned, it is filled with fresh water that usually
cells. SpenceP reported that low light intensity has been treated by chlorination, UV irradiation,
and low salinity promote rapid growth of the or ozone to reduce the number of potential com-
culture. It is necessary to maintain the pH from petitors. Nutrients are then added for algal growth.
6.5 to 8. The starter culture is used to inoculate For autotrophic growth, it is necessary to add a
a step-up production culture, typically 100 to 200 nitrogen source, such as ammonia or nitrate, and
1. The step-up culture under optimal conditions a source of phosphorus. Typically, ammonium
is able to increase from 2 X lo4 to 3 X lo5 in bicarbonate, potassium phosphate, ferric chlo-
about 5 d. ride, and EDTA are added.
Large-scale production is carried out in ponds Huematococcus may also be cultured heter-
containing 30,000 to 1 million 1. Production ponds otrophically. Heterotrophic growth requires an
may be located indoors or outdoors, the indoor organic carbon source, a nitrogen source, phos-
location having the advantage of environmental phorus, and vitamins. Acetate is usually the pre-
control. This is important with Huemutococcus ferred carbon source. Urea is the preferred nitro-
since it is a sweetwater alga and is susceptible gen source, and thiamin is required as a growth
to competition and contamination by other or- factor.
ganisms. Indoor locations are more expensive for In autotrophic culture, the pool is seeded with
production. The outdoor pools are shallow and the step-up culture, usually ranging from 0.5 to
are lined with white plastic. Owing to the poten- 5% of the production volume, but usually 1 to

318
 64

Q) 48
0
c
Q)
0
v)
Q)
5a 32
E
0
0
4
16

-- 4
Forward Scatter
FIGURE 9. FCCS analysis of Phaflia rhodozyma strain 67-385 after rnutagenesis with NTG.
Cells were sorted in windows I and II. See Reference 140 and text for details.

2%. After inoculation, the pool is slowly mixed or shortly thereafter, it is necessary to treat the
with a paddlewheel at one end of the pond. The powder with antioxidants to protect the carot-
pool is gassed with CO, to provide carbon for enoids against degradation. Antioxidants that have
autotrophic growth and to control the pH at 7.3. been used successfully include butylated hydrox-
Best growth is obtained under relatively low light yanisole (BHT), ethoxyquin, tocopherols, pro-
intensities. Growth is complete in about 5 d and pylgallate, and others. These are added at 0.1 to
the cell density usually reaches 3 to 6 X lo5 cells 3% by weight, depending on the antioxidant. Al-
per milliliter. Once this stage is reached, en- ternatively, modified atmosphere packaging can
cystment is encouraged by nutrient deprivation be used but this is usually not preferred since the
and by increasing the salinity of the medium. carotenoids will degrade on storage in the feeds.
Cells are allowed to encyst for an additional 5 d. The cell wall of encysted Huemutococcus is
The nonmotile, thick-walled, red-pigmented en- very tough and difficult to fracture by conven-
cysted cells are then harvested from the pond. tional grinding techniques. Dried cells are more
Mixing is stopped and the cells are allowed to susceptible to fracture in impact and jet mills.
settle. The sedimented cells are collected and Grinding may be carried out at very low tem-
further concentrated by centrifugation or other peratures ( - 50 to - 170"C), which has been re-
means. The paste is often heated to 70°C to con- ported to give an excellent product.
centrate the cells and to inactivate any contam- The carotenoids in the finished product typ-
inating organisms. Further washing and purifi- ically include astaxanthin (primarily esterified),
cation of the cysts may be carried out. a- and @-carotene, lutein, violaxanthin, and
To liberate the pigments, the dried, cleaned neoxanthin. Lesser amounts of other carotenoids,
Haemutococcus cysts are ground to a powder, including free astaxanthin, lutein epoxide, zeax-
having a size of 10 pm or less. During grinding anthin, antheraxanthin, echinenone, canthax-

319
 n

Carotenoid (mglg yeast)

Carotenoid (mg/l medium)

67-385 ( 3 A n t - 1 (32A-2AP (:AAN-4 ( 3 2 A 2 N (3) 2 A 2 N ( 5 ) 2 A 2 N ( 8 : 2 A 2 N (13)

Strain (Day)

FIGURE 10. Carotenoid production by various strains isolated by An116*117

in the
authors' laboratory. Error bars represent one standard deviation

anthin, and other uncharacterized keto-carot- have been described in a European patent appli-
enoids are also included, along with chlorophylls cation. '6.1
a and b. Astaxanthin esters usually comprise 60 Lewis and colleagues have studied various
to 80% of the carotenoid mixture, and ranges production methods for P. rhodozyma with the
from 0.5 to 2% of the dry weight. aim of developing an inexpensive industrial
Production of Huematococcus for astaxan- method of culture. They have successfully cul-
thin appears promising. However, there are still tivated P. rhodozyrna on alfalfa juice'" and have
several problems, including contamination in the evaluated autolysis and mixed culture systems for
sweetwater ponds and liberation of the pigments hydrolysis of the cell wall of P. rhodozyma.165*166
for feed formulation. Although some investigators have experienced
difficulty in culturing Phaffia on molasses,
Haard'67 determined conditions in which molas-
B. Phaffia rhodozyma ses supported good growth and astaxanthin pro-
duction by P. rhodozyma.
Few publications are available concerning in- The culture of P. rhodozyma in fermentors
dustrial cultivation of P. rhodozyma. The yeast is costly compared with other yeast fermentations
is generally grown in stirred-tank reactors and since extended fermentations at low temperatures
controlled for sugar feed and pH. Aeration must are required (up to 5 d). Considerable astaxanthin
be vigorous to obtain good cell mass yields and is also produced after P. rhodozyma growth has
yeast containing high levels of carotenoid. P . stopped. Good cooling capacity and aeration are
rhodozyrna is a durable yeast and its growth is important requirements. Reduction of costs for
not inhibited by vigorous agitation and by CO,. production of P. rhodozyma by cultivation on
Very high dry cell masses have been obtained in sugar or other waste streams could improve the
fermentors (50 to 150 g dry cell mass per liter), economics for yeast astaxanthin.
imparting a spectacular visible impression to an
observer. The fermentations must be provided
with adequate aeration and cooling and must be VIII. CONCLUSION
carried out for several days as the yeast grows
slowly. Preparation of seed cultures, media, etc. The demand for astaxanthin is expected to

320
increase markedly as aquacultural processes are 5. Steven, D. M., Studies on animal carotenoids. I.
developed for farming of prawns, shrimp, lobs- Carotenoids of the brown trout (Salmo trutta Linn.),
J . Exp. Biol., 25, 369, 1948.
ters, and various fishes. The most promising bi- 6. Torrissen, 0. J., Hardy, R. W., and Shearer,
ological sources at present are the alga Haema- K. D., Pigmentation of salmonids -carotenoid dep-
tococcus and the yeast PhafJia rhodozyma. Other osition and metabolism in salmonids, Crit. Rev.
biological sources may be discovered from var- Aquatic Sci., 1, 209, 1989.
ious habitats. Currently the market for astaxan- 7. Bendich, A. and Olson, J. A., Biological actions
of carotenoids, FASEB J . , 3, 1927, 1989.
thin is dominated by the synthetic, but it is pos-
8. Rumsey, G. L., Fish farming as a form of animal
sible that biological sources will begin to compete agriculture: recent advances in fish nutrition, in Proc.
in the 1990s. There are serious limitations for Alltech's 4th Annu. Symp. Biotechnology in the Feed
biological production by algae and yeasts. Eco- Industry, Lyons, T. P . , Ed., Alltech Technical h b -
nomic production of astaxanthin by algae will lications, Nicholasville, KY, 1988, 235.
depend on developments in cultivation to in- 9. Bjorndahl, T., The Economics of Salmon Aquacul-
ture, Blackwell Scientific, Oxford, 1990, 1.
crease the rate of growth and prevent contami- 10. Rackham, D., Salmon and trout marketing, in Salmon
nation in the sweetwater conditions, and devel- and Trout Farming, Laird, L. and Needham, T.,
opment of methods to rupture the cells while Eds., Ellis Horwood, Chichester, England, 1988,231.
maintaining carotenoid stability. Astaxanthin 11. Fox, D. L., The pigments of fishes, in The Physi-
production by P . rhodozyma depends on the iso- ology of Fishes, Vol. 2, Brown, M. E., Ed., Aca-
demic Press, New York, 1957, 367.
lation of carotenoid high-producing strains
12. Kanemitsu, T. and Aoe, H., Studies on carotenoids
(-l0,OOO pg/g) that will stably produce astax- of salmon. I. Identification of the muscle pigments,
anthin in industrial fermentors and perhaps uti- Bull. SOC. Sci. Fish., 24, 209, 1958.
lization of sugar waste streams as inexpensive 13. Kanemitsu, T. and Aoe, H., On the studies of the
growth substrates. Isolation of P. rhodozyma carotenoids of salmon. II. Determination of muscle
strains that grow at temperatures >26"C would pigment, Bull. Jpn. SOC. Sci. Fish., 24, 555, 1958.
14. Khare, A,, MOSS, G. P., Weedon, B. C. L., and
also be beneficial. More research is also neces- Matthews, A. D., Identification of astaxanthin in
sary in fundamental aspects of carotenogenesis, Scottish salmon, Comp.Biochem. Physiol., 45B, 971,
a research area in biotechnology that at present 1973.
deserves considerable attention owing to the rapid 15. Sinnot, R., Fish pigmentation, Trout News, 7, 8 ,
growth in aquaculture. 1988.
16. Mayne, S. T. and Parker, R. S., cis-Canthaxanthin
and other carotenoid-like compounds in canthaxan-
thin preparations, J . Agri. Food Chem., 36, 478,
ACKNOWLEDGMENTS 1988.
17. Storebakken, T., FOSS, P., Austreng, E., and
EAJ thanks Michael J. Lewis, Herman J. Liaaen-Jensen, S., Carotenoids in diets for salmo-
Phaff, and Mary Miranda for introducing him to nids. II. Epimerization studies with astaxanthin in
Phafia rhodozyma. We also thank Danisco Bio- Atlantic salmon, Aquaculture, 44,259, 1984.
technology A / S for current support of our re- 18. Kuhn, R. and Sorenson, N. A., Uber astaxanthin
and ovoerdin, Ber. Dtsch. Chem. Ges., 71, 1879,
search on astaxanthin and various companies and 1983.
Sea Grant for past support. 19. Karrer, P. and Jucker, E., Carotenoids, Elsevier,
Amsterdam, 1950, 239.
20. Andrewes, A. G., Borch, G., Liaaen-Jensen, S.,
and Snatzke, G., Animal carotenoids. IX. On the
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