Báo cáo khoa học: Deviation of the neurosporaxanthin pathway towards b-carotene biosynthesis in Fusarium fujikuroi by a point mutation in the phytoene desaturase gene ppt

Deviation of the neurosporaxanthin pathway towards
b-carotene biosynthesis in Fusarium fujikuroi by a point
mutation in the phytoene desaturase gene
Alfonso Prado-Cabrero
1
, Patrick Schaub
2
, Violeta Dı
´
az-Sa
´
nchez
1
, Alejandro F. Estrada
1
,
Salim Al-Babili
2
and Javier Avalos
1
1 Departamento de Gene
´
tica, Universidad de Sevilla, Spain
2 Albert-Ludwigs University of Freiburg, Faculty of Biology, Germany
Introduction
Carotenoids are terpenoid pigments widely distributed
in nature, produced by all photosynthetic organisms
[1] and many nonphotosynthetic microorganisms, such
as bacteria and fungi [2,3]. In plants and algae, carote-
noids play essential roles as accessory pigments in pho-
tosynthesis [4], and provide red, orange, or yellow

colours to many fruits and flowers. Animals lack the
ability to synthesize carotenoids and rely on their diet
to produce the vision chromophore retinal [5] or the
vertebrate morphogen retinoic acid [6]. Carotenoids
are also beneficial for human health as protective
agents against oxidative stress, cancer, sight degenera-
Keywords
carB; carotenogenesis; carotenoid
overproducing mutant; filamentous fungi;
PDS enzyme
Correspondence
J. Avalos, Departamento de Gene
´
tica,
Universidad de Sevilla, Apartado 1095,
E-41080 Sevilla, Spain
Fax: +34 95 455 7104
Tel: +34 95 455 7110
E-mail:
(Received 12 May 2009, revised 12 June
2009, accepted 22 June 2009)
doi:10.1111/j.1742-4658.2009.07164.x
Carotenoids are widespread terpenoid pigments with applications in the
food and feed industries. Upon illumination, the gibberellin-producing fun-
gus Fusarium fujikuroi (Gibberella fujikuroi mating population C) develops
an orange pigmentation caused by an accumulation of the carboxylic apoc-
arotenoid neurosporaxanthin. The synthesis of this xanthophyll includes
five desaturation steps presumed to be catalysed by the carB-encoded phy-
toene desaturase. In this study, we identified a yellow mutant (SF21) by
mutagenesis of a carotenoid-overproducing strain. HPLC analyses indi-

cated a specific impairment in the ability of SF21-CarB to perform the fifth
desaturation, as implied by the accumulation of c-carotene and b-carotene,
which arise through four-step desaturation. Sequencing of the SF21 carB
allele revealed a single mutation resulting in an exchange of a residue con-
served in other five-step desaturases. Targeted carB allele replacement
proved that this single mutation is the cause of the SF21 carotenoid pat-
tern. In support, expression of SF21 CarB in engineered carotene-produc-
ing Escherichia coli strains demonstrated its reduced ability to catalyse the
fifth desaturation step on both monocyclic and acyclic substrates. Further
mutagenesis of SF21 led to the isolation of two mutants, SF73 and SF98,
showing low desaturase activities, which mediated only two desaturation
steps, resulting in accumulation of the intermediate f-carotene at low levels.
Both strains contained an additional mutation affecting a CarB domain
tentatively associated with carotenoid binding. SF21 exhibited higher carot-
enoid amounts than its precursor strain or the SF73 and SF98 mutants,
although carotenogenic mRNA levels were similar in the four strains.
Abbreviations
PDS, phytotene desaturase; PPO, protoporphyrinogen IX oxidase.
4582 FEBS Journal 276 (2009) 4582–4597 ª 2009 The Authors Journal compilation ª 2009 FEBS
tion syndromes and cardiovascular diseases [7]. In
addition, carotenoids are responsible for the pigmenta-
tion of some birds, insects, fish and crustaceans.
Most naturally occurring carotenoids share a typical
chemical structure derived from the C
40
polyene chain
of the colourless precursor phytoene, a carotene syn-
thesized by the enzyme phytoene synthase through the
condensation of two geranylgeranyl pyrophosphate
molecules (Fig. 1). Carotenoid biosynthetic pathways

proceed through the sequential introduction of conju-
gated double bonds in the phytoene backbone to yield
increasingly desaturated molecules absorbing visible
light. Desaturation steps are usually followed by cycli-
zation reactions catalysed by carotene cyclases. The
generated end-rings may be further modified by differ-
ent oxidases introducing oxygen-containing functional
groups. Carotenoids are divided into carotenes consist-
ing of hydrocarbons and their oxygenated derivatives
the xanthophylls [8].
Desaturation steps are achieved by a group of
enzymes, with phytoene desaturases (PDSs) as their
most representative members. PDS enzymes differ in
the number of introduced double bonds, which range
from two to five [9]. Some PDS-related enzymes desat-
urate substrates other than phytoene, e.g. hydroxyneu-
rosporene [10], dehydrosqualene [11] or f-carotene [12].
Plants, algae and cyanobacteria employ two enzymes,
PDS and f-carotene desaturase, to perform the four
desaturation reactions required for lycopene formation
[13]. These enzymes are evolutionarily related to each
other and to the hydroxyneurosporene dehydrogenase
of Rhodobacter sphaeroides [10], but show low
sequence similarity to other bacterial counterparts like
the Pantoea phytoene desaturase CrtI. The low
sequence conservation suggests a convergent evolution
of both groups, further substantiated by their different
sensitivities to chemical inhibitors [9]. Other PDS-
related enzymes act as isomerases [14], e.g. the plant
and cyanobacterial prolycopene isomerase CrtISO [15],

or as saturases, e.g. the animal all-trans-retinol:all-
trans-13,14-dihydroretinol saturase RetSat [16].
Many fungal species are useful tools for the produc-
tion of secondary metabolites and the analysis of their
biosyntheses. One example is the ascomycete Fusari-
um fujikuroi (Gibberella fujikuroi MP-C), known for its
ability to produce gibberellins [17], growth-promoting
plant hormones with agricultural applications. Upon
illumination, F. fujikuroi develops an orange pigmenta-
tion caused by the accumulation of neurosporaxanthin
[18], a carboxylic apocarotenoid originally found in the
fungus Neurospora crassa [19]. Neurosporaxanthin is
produced from phytoene through five desaturations,
an end-cyclization, an oxidative cleavage reaction and
a final oxidation step (Fig. 1). This pathway is medi-
ated by the PDS CarB [20,21], the bifunctional phyto-
ene synthase ⁄ carotene cyclase CarRA [21], the
carotenoid cleaving oxygenase CarT [22] and finally by
the presumed aldehyde dehydrogenase CarD, which is
currently under investigation. F. fujikuroi also accumu-
lates minor amounts of b-carotene [18] resulting from
Fig. 1. Carotenoid and retinal biosynthesis
in Fusarium fujikuroi. The pathway involves
CarRA, CarB, the cleaving oxygenases CarX
and CarT, and a postulated dehydrogenase
CarD. Desaturations introduced by the CarB
enzyme are circled. The grey arrow indi-
cates the reaction affected in the SF21
mutant. Reactions under-represented in this
strain are shaded.

A. Prado-Cabrero et al. Alteration of Fusarium phytoene desaturase
FEBS Journal 276 (2009) 4582–4597 ª 2009 The Authors Journal compilation ª 2009 FEBS 4583
end-cyclization of the intermediate c-carotene, cataly-
sed by CarRA (Fig. 1). b-Carotene is the substrate for
CarX, a second carotenoid-cleaving oxygenase, which
produces retinal [23,24]. Expression of the identified
car genes is stimulated by light and derepressed in the
dark in carotenoid-overproducing mutants, generically
called carS [22,23,25]. The mutated regulatory gene(s)
responsible for the carS phenotype remains to be iden-
tified.
As in F. fujikuroi, a single desaturase gene has been
found in other carotenogenic fungi: the ascomycetes
N. crassa (al-1) [26] and Cercospora nicotianae (pdh1)
[27], the zygomycetes Phycomyces blakesleeanus, Mu-
cor circinelloides and Blakeslea trispora (carB) [28–30]
and the basidiomycete Xhanthophyllomyces dendror-
hous (crtI) [31], formerly Phaffia rhodozyma. These
enzymes, more similar to those of carotenogenic bacte-
ria than to desaturases of photosynthetic organisms,
are presumably responsible for all desaturation steps
in the corresponding carotenoid pathways. The ability
to carry out four desaturations was first inferred from
genetic approaches for the CarB PDS from P. blakes-
leeanus [32], and later confirmed by heterologous
expression in Escherichia coli [28]. A similar heterolo-
gous approach demonstrated the ability of CrtI from
X. dendrorhous to catalyse the four desaturations from
phytoene to lycopene [31] and of AL-1 from N. crassa
to achieve the five desaturations from phytoene to 3,4-

didehydrolycopene [33]. The carotenoid pathway of
N. crassa coincides with that of F. fujikuroi in the syn-
thesis of the same end-product, the apocarotenoid
neurosporaxanthin, but both fungi differ in the order
of the reactions. Whereas in N. crassa the five desatu-
rations are performed first and followed by cyclization
reaction as a later step [34], in F. fujikuroi the cycliza-
tion reaction precedes the fourth and fifth desatura-
tion steps, as indicated by the absence of lycopene
and the occurrence of b-zeacarotene in different
strains [18].
The accumulation of phytoene in carB mutants [20]
and the lack of strains blocked in a later desaturation
step indicated that CarB is responsible for all five
desaturations. Here, we provide conclusive evidence for
the ability of the CarB enzyme to carry out the five
desaturation reactions and to discriminate between
different carotenoid substrates. We have isolated and
characterized a F. fujikuroi carB mutant impaired in
the catalysis of the fifth desaturation (i.e. that con-
verting c-carotene to torulene), but fully able to cata-
lyse the preceding four desaturation steps. The effect
of the mutation was confirmed by targeted allele
replacement and comparing the activity of wild-type
and altered CarB enzymes in different carotenoid-
producing E. coli strains. Finally, a hypothesis is
proposed to explain the structural basis of the effect
of the mutation.
Results
Isolation and phenotypic analysis of a yellow

mutant
The pale pigmentation of wild-type F. fujikuroi hinders
the identification of colour mutants with alterations in
the carotenoid pattern. Such mutants are easily identi-
fied in deeply orange-pigmented strains like the carS
carotenoid-overproducing mutants [18]. A screening
for colour mutants was performed after chemical
mutagenesis of the carS strain SF4, a descendent of
the nitrate reductase-deficient mutant SF1 (Table 1),
not affected in carotenoid biosynthesis and formerly
used as a recipient strain for transformation experi-
ments [25]. This search led to the identification of a
mutant with a striking yellow colour (Fig. 2). This
mutant was subcultured from single conidia and
denominated as SF21.
The carotenoids produced by SF21 were analysed
by spectrophotometry, TLC and HPLC (Fig. 2). As
indicated by their colours, UV ⁄ Vis spectra of the
SF21 carotenoid samples differed from that of its
ancestor strain SF4 in shape and maximal absorption.
TLC analyses revealed that most of the carotenoids
accumulated by SF4 were highly polar, pointing to
neurosporaxanthin as the predominant component.
The minor neutral fraction contained torulene and
traces of other carotene intermediates. Parallel separa-
tion of the SF21 crude carotenoid samples revealed
two predominant bands corresponding to c-carotene
and b-carotene. In contrast to SF4, no torulene could
be detected, and the neurosporaxanthin band was
much paler. HPLC analyses of the neutral carotenoid

fractions from both strains confirmed the predomi-
nance of torulene in SF4 and the accumulation of
large amounts of c-carotene and b-carotene in SF21
(Fig. 2). The amount of phytoene was low in both
strains, but was significantly higher in SF21 than in
SF4.
Quantification of the carotenoid contents in mycelial
samples from light- or dark-grown cultures showed
similar results, except for a higher neurosporaxanthin
content in the illuminated SF4 samples (Fig. 2). As
expected, its parental strain SF1 produced moderate
amounts of neurosporaxanthin only in the light. The
carotenoid concentration was at least threefold higher
in SF21 than in SF4, with neurosporaxanthin repre-
senting < 10% of the total carotene.
Alteration of Fusarium phytoene desaturase A. Prado-Cabrero et al.
4584 FEBS Journal 276 (2009) 4582–4597 ª 2009 The Authors Journal compilation ª 2009 FEBS
Identification of a mutation in the SF21 carB
allele
The carotenoid pattern of the mutant SF21, i.e. the
accumulation of c-carotene and the subsequent
deviation of the pathway to b-carotene, suggested
impaired c-carotene to torulene desaturation activity.
This may be the result of an altered CarB if all five
desaturations required for torulene synthesis are
catalysed by this sole F. fujikuroi PDS enzyme
(Fig. 1). To test this hypothesis, we cloned and
sequenced the carB alleles from strains SF21 and
wild-type FKMC1995.
The carB gene was formerly cloned from the wild-

type F. fujikuroi strain IMI58289 [21] and its sequence
was deposited in the EMBL database (accession num-
ber AJ426418). The carB sequence from FKMC1995
was identical to that of IMI58289 except for a
C196 fi T transition which does not affect the
encoded protein sequence. The predicted CarB protein
shared a similar structural organization with other
PDS and PDS-related enzymes of different origins
(Fig. 3), including the characteristic N-terminal dinu-
cleotide-binding domain [35,36].
Compared with carB from FKMC1995, the SF21
carB allele, designated here as carB36, showed a single
point mutation, a C608 fi T transition, resulting in a
Pro170 fi Leu substitution. The corresponding resi-
due is located in a predicted a-helix-rich region
(Fig. 3) far from the presumed carotene binding
domain harbouring the mutations formerly identified
in three P. blakesleaanus carB mutants [37].
Replacement of the wild-type carB allele by
carB36
The generation of mutant SF21 from wild-type
FKMC1995 includes two chemical mutagenesis steps
(Table 1), presumably resulting in further random
mutations in addition to that found in the SF21
carB36 allele. To check if this allele is sufficient to pro-
duce the deviation of the pathway to b-carotene in a
wild-type carotenogenesis background, a two-step
strategy was used to replace the carB allele of strain
SF1 with carB36 (Fig. 4A,C). Ten transformants were
isolated after transformation of the SF1 strain with a

plasmid carrying carB36. In five of them, Southern
blot analyses showed the incorporation of a single
copy of the plasmid at the carB locus (Fig. 4A,B).
Three of these strains were checked for carotenoid
content. Compared with the wild-type, the three trans-
formants contained approximately twofold more carot-
enoids upon illumination, but exhibited similar
carotenoid compositions. One of these transformants
(T5, indicated by an asterisk in Fig. 4B) was chosen
for further investigation. T5 conidia were grown on
Petri dishes to search for mutant colonies, expected at
low frequency from spontaneous plasmid loss by
homologous recombination (Fig. 4C). Transfer of indi-
vidual colonies to selective medium showed variable
frequencies of hygromycin sensitive strains, usually
> 1%. However, all the strains tested were orange
and contained the wild-type carB allele, suggesting
preferential recombination through the same DNA
segment that led to the plasmid integration. No yellow
Table 1. Fusarium fujikuroi strains used in this study. Only the relevant transformant is included. For clarity, wild-type carB alleles (carB
+
)
are also indicated. NG, N-methyl-N¢-nitro-N-nitrosoguanidine.
Strain Genotype
a,b
Origin Colour in the dark
FKMC1995 carB
+
White
SF1 niaD4 carB

+
FKMC1995, spontaneous
ClO
3
K resistance
White
SF4 niaD4 carS35 carB
+
SF1, NG mutagenesis Orange
SF21 niaD4 carS35 carB36 SF4, NG mutagenesis Yellow
SF73 niaD4 carS35 carB37 SF21, NG mutagenesis Greenish
SF98 niaD4 carS35 carB38 SF21, NG mutagenesis Pale greenish
T5 niaD4 carB
+
⁄ carB36 hygR SF1, transformation with pB21H White
SF191 niaD4 carS63 carB
+
⁄ carB36 hygR T5, NG mutagenesis Orange
SF214 niaD4 carS63 carB36 SF191, spontaneous
plasmid loss
Yellow
SF215 niaD4 carS63 carB36 SF191, spontaneous
plasmid loss
Yellow
SF216 niaD4 carS63 carB
+
SF191, spontaneous
plasmid loss
Orange
a

carS mutations are tentatively assigned to a single hypothetical carS gene.
b
carB37 and carB38 alleles include also the carB36 mutation.
A. Prado-Cabrero et al. Alteration of Fusarium phytoene desaturase
FEBS Journal 276 (2009) 4582–4597 ª 2009 The Authors Journal compilation ª 2009 FEBS 4585
colonies were detected after visual inspection of at
least 120 Petri dishes with  250–500 colonies, proba-
bly because of the difficult identification of this pheno-
type in the pale pigmented background of T5.
Because SF21 was obtained from a carotenoid-over-
producing strain, a mutagenesis experiment was used
to obtain a T5-derived carS mutant, termed here
SF191. This deregulated strain contained more carote-
noids than SF4 (Fig. 5A), as expected from the pres-
ence of two carB genes, one with the carB36 mutation
(Fig. 4A). Hygromycin-sensitive strains were obtained
from SF191 and checked by PCR for the loss of the
carB36 allele. One of them, called SF216, harboured a
single wild-type carB allele (PCR test not shown) and
had a lower carotenoid content (Fig. 5A) than SF191.
In contrast to SF4, SF216 contained similar amounts
of carotenoids in dark or light, indicating differences
in their respective carS mutations.
Conidia collected from SF191 were grown in the
same media and screened for the generation of yellow
Fig. 2. SF21 phenotype. Representative colonies of SF4 and SF21 strains grown in the dark at 22 °C on DGasn agar. TLC and HPLC analy-
ses of carotenoid samples from 9-day-old mycelia of both strains grown under the same conditions. UV ⁄ Vis spectra (350–550 nm) and maxi-
mal absorbance wavelengths (nm) of accumulated carotenoids are shown in the insets. Below: quantitative analyses of the carotenoids
produced by SF1, SF4 and SF21. A scheme of the pathway is presented on the left. Phytoene, phytofluene, f-carotene, b-zeacarotene, c-car-
otene, b-carotene, torulene and neurosporaxanthin are abbreviated as P, Pf, f, b-z, c, b, T and Nx, respectively. The identities of the interme-

diates are depicted by colour. Surfaces are proportional to amounts, indicated in lgÆg
)1
dry mass. The data show average and standard
deviation (outer semicircles) from three independent determinations. Left and right semicircles correspond to cultures grown in the dark and
under continuous light, respectively. SF1 contained only trace amounts of carotenoids in the dark. SF4 contained low amounts of phytoene,
c-carotene and b-carotene, represented as approximate calculations. Circles missing in the SF4 and SF21 schemes correspond to undetected
carotenoids.
Alteration of Fusarium phytoene desaturase A. Prado-Cabrero et al.
4586 FEBS Journal 276 (2009) 4582–4597 ª 2009 The Authors Journal compilation ª 2009 FEBS
colonies. Two yellow colonies were identified in a
screening of  5000; both strains, called SF214 and
SF215, were sensitive to hygromycin, indicating loss of
the integrated plasmid. As predicted, both mutants
lacked the FokI restriction site, present in the wild-type
carB allele, but not in the carB36 mutant allele
(Fig. 4D), confirming the expected allele replacement.
Like SF21, SF214 and SF215 contained low amounts
of neurosporaxanthin. Moreover, HPLC analyses of
their neutral carotenoid fractions showed a pattern
very similar to that of SF21 either in dark- or in light-
grown cultures (Fig. 5B). This result strongly indicated
that the carB36 mutation is responsible for the yellow
phenotype, i.e. the defective CarB capacity to carry
out the fifth desaturation step in the neurosporaxan-
thin biosynthetic pathway.
Heterologous expression of the carB36 allele
To further confirm the effect of the carB36 mutation
on enzyme activity, wild-type carB and carB36 cDNAs
were cloned and expressed in different carotene-pro-
ducing E. coli strains and the resulting carotene

patterns were determined (Fig. 6). Expression of wild-
type carB in a phytoene producing E. coli strain
resulted in an efficient desaturation to lycopene accom-
panied by a lower production of 3,4-didehydrolycopene,
indicating that the fifth desaturation is less efficiently
achieved than the preceding four in the E. coli back-
ground. Expression of carB36 resulted in lycopene
amounts at least comparable with those formed by
CarB, whereas the production of 3-4 didehydrolyco-
pene was reduced approximately sixfold (Fig. 6A).
Similar results were obtained through introducing the
two cDNAs in a lycopene-producing E. coli strain,
expressing the bacterial four-step desaturase gene crtI.
As shown in Fig. 6B, the activities of CarB and
CarB36 led to similar lycopene contents, whereas the
amounts of 3,4-didehydrolycopene were approximately
eightfold higher in the carB-expressing strain.
The carotenoid pattern of F. fujikuroi indicates that
the substrate for the fifth desaturation step is c-caro-
tene rather than lycopene. Therefore, we expressed the
two cDNAs in a c-carotene-accumulating E. coli
strain, engineered by introduction of the bacterial
desaturase CrtI and the N. crassa cyclase ⁄ phytoene
synthase AL-2 [34]. Compared with CarB36, the activ-
ity of CarB led to a sevenfold higher quantity of toru-
lene (Fig. 6C). Similarly, the conversion of lycopene to
3,4-didehydrolycopene, achieved in parallel in the same
cells, was much higher in CarB-expressing cells. Taken
together, the usage of the E. coli system confirmed the
specific effect of the carB36 mutation on the fifth

desaturation reaction.
Fig. 3. Alignment of predicted structures for phytoene desaturases from the fungi Fusarium fujikuroi (CarB Ff, accession number
CAD19989.2), Neurospora crassa (AL-1 Nc, XP964713), Xhanthophyllomyces dendrorhous (CrtI Xd, AAO53257) and Phycomyces blakeslee-
anus (CarB Pb, CAA55197.1), the bacteria Rhodobacter sphaeroides (CrtI Rs, YP353345), the archaea Sulfolobus solfataricus (CrtI Ss,
NP344226), and the plant Arabidopsis thaliana (PDS3 At, Q07356). The comparison also includes the A. thaliana f-carotene desaturase
(ZDS1 At, Q38893) and the human all-trans-retinol 13,14-reductase (RetSat Hs, Q6NUM9). Structures were deduced with the program
3D-PSSM. Broad rectangles represent predicted a helices, and thin rectangles represent predicted b sheets. The conserved b–a–b dinucleo-
tide-binding domain is indicated in black. The a-helix-rich segment is shaded in grey. Polarity of the helices of this region and the presence
of basic residues in the F. fujikuroi enzyme are indicated (
•
,hydrophilic; , moderately hydrophobic; s, highly hydrophobic; each short line
below is either a lysine or an arginine residue). Vertical lines and arrows indicate mutations; SF21, Pro170 fi Leu; SF73, Trp449 fi Stop;
SF98, Gly504 fi Asp (described in this work); A486, Glu426 fi Lys; C5, Ser444 fi Phe and Leu446 fi Phe; S442, Glu482 fi Lys,
described by Sanz et al. [37]. The asterisk marks the mutation in the R. sphaeroides PDS enzyme that provides the ability to carry out a
fourth desaturation.
A. Prado-Cabrero et al. Alteration of Fusarium phytoene desaturase
FEBS Journal 276 (2009) 4582–4597 ª 2009 The Authors Journal compilation ª 2009 FEBS 4587
Further alterations in carB activity
To determine further residues essential for other desat-
uration steps, mutagenesis experiments of the SF21
strain were performed, leading to the isolation of low-
pigmented strains. Two of them were SF73 and SF98,
which exhibited a pale greenish hue. HPLC analyses of
these two mutants revealed the accumulation of phyto-
ene and lower amounts of f-carotene (Fig. 7). SF98
also contained minor amounts of c-carotene and b-car-
otene, whereas SF73 was hardly able to desaturate
f-carotene. These SF73 and SF98 carotene patterns
suggested the occurrence of further mutations in the
carB gene, resulting in more impaired PDSs.

Sequence analysis of the corresponding carB genes
showed a G1493 fi A transition in the SF73 carB
allele, resulting in a Trp449 fi Stop mutation. The
predicted truncated protein lacks the C-terminal 121
amino acids, which include the putative carotene-bind-
ing domain [37], making the accumulation of minor
amounts of carotenoids an unexpected result. The
SF98 carB allele contains a G1657 fi A transition,
leading to a Gly504 fi Asp replacement in the caro-
tene-binding domain (Fig. 3). The phenotype of the
SF73 and SF98 mutants could be also caused by a
combined effect of these mutations with the carB36
Pro170 fi Leu substitution, also present in these
strains.
Relation between carotenoid biosynthesis and
expression of the car genes in carS and carS
⁄
carB
mutants
As shown in Figs 7 and 8, the striking difference in
the carotenoid content between SF21 and its precursor
strain SF4 was less pronounced in the SF21-derived
A
BD
C
Fig. 4. carB allele replacement. (A) Physical map of the pB21H integration at the homologous carB sequence by a single recombination
event in the genome of strain SF1. The mutation in the carB36 allele is indicated by a star. The carB probe, relevant BamHI sites used for
Southern blot analyses and expected fragment sizes are indicated. (B) Southern blot analyses of the recipient strain SF1 and 10 transfor-
mants. Squares highlight transformants whose hybridization pattern indicates the incorporation of a single copy of the plasmid at the carB
locus. The transformant T5 was used in further experimental steps. (C) Physical map of molecular events leading to loss of the plasmid

pB21H by a single recombination at the homologous carB sequence in the genome of a carS strain derived from T5. The recombination
shown occurs at the opposite side from the one that produced the plasmid integration, leaving the mutated carB allele in the genome. (D)
Electrophoretic profiles of the PCR products obtained with primers flanking the mutation site at allele carB36 using DNA from wild-type,
SF21, SF191, SF214 and SF215 strains and digested with FokI. Interpretation of expected bands is depicted on the right scheme. The SF21
mutation leads to the loss of a FokI restriction site.
Alteration of Fusarium phytoene desaturase A. Prado-Cabrero et al.
4588 FEBS Journal 276 (2009) 4582–4597 ª 2009 The Authors Journal compilation ª 2009 FEBS
mutants SF73 and SF98. Furthermore, the latter
mutants regained the light induction of carotenogene-
sis, which had disappeared in the parental strain SF21
(Fig. 8). To check whether the differences in carotene
content are a result of altered mRNA levels for the
carotenogenic enzymes, we carried out northern blot
experiments.
As expected, carRA or carB mRNAs were undetect-
able in dark-grown mycelia of the wild-type and SF1
strains and highly induced after 1 h exposure to light
BA
Fig. 5. Effect of wild-type and carB36 alleles on Fusarium fujikuroi carotenogenesis. (A) Carotenoids accumulated by the mutants SF1, SF4,
SF21, SF191 and SF216. (B) Neutral carotenoids accumulated by the mutants SF21, SF214 and SF215. The analyses were carried out on
mycelial samples from dark or light-grown cultures (5 WÆm
)2
). The data show average of two independent experiments.
ABC
Fig. 6. CarB36 desaturation activity of CarB and CarB36 in Escherichia coli strains producing different carotene substrates. Based on HPLC
analyses, the data show carotenoid compositions of three E. coli strains accumulating different carotenoid intermediates and expressing
wild-type thioredoxin-carB,-carB36 or thioredoxin (control). The three E. coli strains were engineered by introducing the following enzymes:
(A) phytoene synthase (phytoene accumulation in the control); (B) phytoene synthase and the bacterial desaturase CrtI (lycopene accumula-
tion); (C) phytoene synthase, CrtI and the Neurospora phytoene synthase ⁄ lycopene cyclase AL-2 (lycopene and c-carotene accumulation).
The data show average and standard deviation of three independent experiments. 3,4ddl = 3,4-didehydrolycopene.

A. Prado-Cabrero et al. Alteration of Fusarium phytoene desaturase
FEBS Journal 276 (2009) 4582–4597 ª 2009 The Authors Journal compilation ª 2009 FEBS 4589
(Fig. 8). mRNA levels in dark-grown SF4 were similar
to those of illuminated wild-type and SF1 strains and
exhibited a significant increase because of light expo-
sure, correlating with enhanced carotenoid production.
Similar expression patterns were observed for SF21,
SF73 and SF98, indicating that the SF21 increased
carotenoid content is not caused by enhanced tran-
script levels.
Discussion
The carotenoid biosynthetic pathway of the orange-
pigmented F. fujikuroi includes a sequence of five
desaturation steps, consisting of two pairs of equivalent
reactions at symmetrical sites in the carotene skeleton,
predictably interrupted by a cyclization of the interme-
diate neurosporene and completed by a fifth reaction
in an outer position to produce torulene (Fig. 1). We
have identified a yellow-pigmented mutant, SF21,
exhibiting a novel carotenoid pattern. Consistent with
its deep yellow colour, SF21 accumulates large
amounts of c- and b-carotene and minor amounts of
the final product neurosporaxanthin, indicating a
specific defect on the CarB ability to catalyse the fifth
desaturation reaction. Previous studies indicated that
CarB is responsible for all desaturation steps of the
pathway. Lack of mutants whose end product is any
of the partially desaturated intermediates was inter-
preted as an indication of the achievement of the five
reactions by a single desaturase [18]. The investigations

of the carB36 allele and the encoded enzyme, reported
here, provide solid support to this assumption. More-
over, our results show that CarB36 desaturase is
unique in the specific impairment of the fifth desatura-
tion reaction, which is caused by a single amino acid
exchange in the wild-type enzyme.
CarB shares a similar structural organization with
other PDS enzymes, as revealed by our secondary
structure predictions using the 3d-pssm protein-fold
recognition program [38]. The same overall structure,
including the b–a–b dinucleotide-binding domain
[35,36], is displayed by phylogenetically distant PDS-
related enzymes like the f-carotene desaturase from
Arabidopsis thaliana, which shows only 14% sequence
identity to CarB. Several carB mutations formerly
investigated in the zygomycete P. blakesleeanus are
located in a region close to the carboxy-end of the pro-
tein [37], tentatively associated with binding of the
carotenoid substrate [39]. One of these mutants, S442,
exhibits a defective PDS with partial activity for the
first two desaturations, leading to the accumulation of
significant amounts of f-carotene [40]. In this study,
we identified two pale greenish strains, the SF21-
derived mutants SF73 and SF98, exhibiting a pheno-
type similar to that of the P. blakesleeanus S442 caused
by mutations affecting the same protein domain.
The different carotenoid patterns of SF73 and SF98
reflect defective PDSs maintaining certain activities
with respect to the first pair of reactions but different
capacities to perform the second pair of desaturations.

The leaky activity of the SF98 desaturase resulted in
the accumulation of significant amounts of f-carotene
and c-carotene. However, we could not detect their
respective precursors in the pathway, i.e. phytofluene
or b-zeacarotene, indicating that when a desaturation
reaction occurs, the symmetric reaction is readily
achieved. A similar result was found with the mutant
SF73, possessing a more severely impaired desaturase,
Fig. 7. Scheme of the carotenoids accumulated by mutants SF21, SF73 and SF98 under continuous illumination. The pathway on the left
includes only detected carotenoids. Phytoene, f-carotene, c-carotene, b-carotene, and neurosporaxanthin are abbreviated as P, f, c, b and
Nx, respectively. Circle surfaces are proportional to carotenoid amounts, indicated in lgÆg
)1
dry mass.
Alteration of Fusarium phytoene desaturase A. Prado-Cabrero et al.
4590 FEBS Journal 276 (2009) 4582–4597 ª 2009 The Authors Journal compilation ª 2009 FEBS
which lost the ability to carry out the second pair of
desaturations but maintained a low, but significant,
capacity to produce f-carotene. However, despite its
low desaturating activity, no phytofluene was accumu-
lated. Interestingly, the SF73 desaturase represents a
truncated CarB lacking the C-terminus, which includes
the presumed substrate-binding domain. Hence, partic-
ipation of other protein segments in carotene binding
must be concluded.
The carB36 mutation is located in a predicted
a-helix-rich protein domain, apparently distant from
the carboxy domain formerly interpreted as involved
in carotene binding. A single mutation in the same a-
helix-rich domain of the three-step PDS of Rhodobacter
sphaeroides (Fig. 3) allows this enzyme to recognize

neurosporene as a substrate [41], supporting a relevant
role for this PDS segment in substrate recognition.
This a-helix-rich domain is similar to the proposed
membrane surface-binding domain of protoporphyri-
nogen IX oxidase (PPO) [42], an enzyme structurally
related to PDSs. The PPO domain is characterized by
the presence of amphipathic a helices rich in basic
amino acids that interact with the phospholipid head
groups of the lipid bilayer, embedding partially into
the membrane and constituting a pore, which enables
entrance of the hydrophobic substrate. As in other
organisms, fungal PDSs are membrane-bound proteins
[43,44] that act on hydrophobic substrates occurring in
the lipid bilayer. The a helices of the PDS domain
mentioned above have different hydrophobicities, and
five of them contain basic amino acids that could
interact with phospholipid head groups (Fig. 3). PDS
enzymes might employ a membrane-binding and sub-
strate-uptake mechanism similar to that of PPO. The
Fusarium carB36 mutation could alter the conforma-
tion of a putative pore, preventing the recognition
and ⁄ or entrance of c-carotene.
The proline residue replaced in the predicted F. fu-
jikuroi CarB36 protein is found in the PDSs AL-1
from N. crassa and CrtI from X. dendrorhous, presum-
ably able to carry out five desaturations [33,45,46].
Conversely, the PDSs from the b-carotene-producing
zygomycetes M. circinelloides, P. blakesleeanus and
B. trispora, which carry out only four desaturations,
contain an aliphatic residue instead of proline at the

same position. However, this rule seems not to be
valid for the PDS of the ascomycete C. nicotiane,
described as producing b-carotene [27], because this
enzyme is highly similar to CarB from F. fujikuroi
( 70% identical amino acids), including the con-
served proline residue. Carotene analysis of the close
relative C. cruenta shows different carotenoids, but
none of them result from a fifth desaturation [47].
Based on our observations, a side branch of the
carotenoid pathway in C. nicotiane involving a fifth
desaturation cannot be discarded. This is actually the
case in X. dendrorhous, where the four-desaturation
pathway into b-carotene and astaxanthin coexists with
a lateral production of torulene [45,46]. The preva-
lence of astaxanthin biosynthesis implies a highly effi-
cient cyclase activity, which competes with the fifth
desaturation step.
Former studies proposed a mechanism of action for
fungal PDSs organized as oligomers. In P. blakeslee-
Fig. 8. Total carotenoid contents and mRNA levels for genes carRA
and carB in the wild-type and the mutants SF1, SF4, SF21, SF73
and SF98. D: grown in the dark. L: grown under continuous illumi-
nation. Northern blot analyses were performed with total RNA sam-
ples. rRNA bands are shown below each panel as load controls.
The bars below each northern blot show the ratios of signal intensi-
ties to rRNA controls; the values are expressed relative to the
maximum in each panel, taken as 1.
A. Prado-Cabrero et al. Alteration of Fusarium phytoene desaturase
FEBS Journal 276 (2009) 4582–4597 ª 2009 The Authors Journal compilation ª 2009 FEBS 4591
anus, each PDS monomer carries out a single desatura-

tion and transfers the desaturated product to the next
monomer, the process being repeated up to the accom-
plishment of four desaturation steps [32,37]. A similar
complex was proposed for the AL-1 PDS of N. crassa,
but in this case the complex would be able to incorpo-
rate partially desaturated carotenoids, either natural
intermediates or unusual hydoxylated substrates [33].
The incorporation of different carotenoids was ele-
gantly demonstrated with a bacterial PDS, which
proved able to introduce further desaturations in
unnaturally long carotenoid substrates [48]. In the case
of the F. fujikuroi CarB, the identification of b-zeacar-
otene implies the release of neurosporene by an even-
tual CarB complex and the latter incorporation of the
cycled substrate. Some PDS-related enzymes work as
homodimers, in which each monomer carries out all
the desaturations. A well-known example is provided
by PPO, which introduces six desaturations in proto-
porphyrinogen [49]. This may be also the case of the
Fusarium CarB enzyme. If b-zeacarotene and c-caro-
tene are incorporated independently by the CarB
enzyme, CarB36 could then be impaired in the ability
to incorporate c-carotene rather than in the desaturase
activity.
The total amount of carotenoids accumulated by
SF21 is at least three times higher than in its neuros-
poraxanthin-overproducing precursor strain SF4,
although their carRA and carB mRNA levels remain
unaltered. The amount of carotenoids in vivo is pre-
sumably determined by the balance between biosyn-

thetic activities and carotenoid degradation rates.
Therefore, the higher carotenoid content in SF21 may
be explained either by a higher activity or the CarB36
enzyme compared with the wild-type counterpart or by
a higher stability of c-carotene and b-carotene com-
pared with torulene and neurosporaxanthin. This may
actually be the case for torulene, as indicated by the
fast decoloration of a torulene-accumulating mutant
upon aging (unpublished observation). The decreased
accumulation of carotenoids in the carS mutant with
two carB genes – wild-type and carB36 – compared
with the SF21 mutant (Fig. 5) supports the hypothesis
on differential carotenoid stability.
The c-carotene and b-carotene accumulation result-
ing from the carB36 mutation may have biotechnologi-
cal implications in other species. For example,
replacement of the carB wild-type allele by carB36 in
Fusarium venenatum, a fungus used by the food indus-
try as the source for mycoprotein [50], may result in
the predominant synthesis of c-carotene and b-caro-
tene and give added value to mycoprotein for human
consumption. Furthermore, a similar mutation in
X. dendrorhous could deviate the biosynthesis further
toward b-carotene and, therefore, favour astaxanthin
production, which is used by the aquaculture feed
industry to provide colour to salmon and crustaceans
[51]. As key enzymes in their biosynthesis, PDSs rep-
resent obvious targets for improved biotechnological
production of carotenoids. E. coli and yeast cells
have been engineered to produce industrially relevant

carotenoids [52-54], and special attention has been
paid to PDSs, whose desaturation activities have
been altered by molecular breeding and directed evo-
lution [41,55]. Moreover, the bacterial PDS CrtI was
employed to generate transgenic plants accumulating
b-carotene, such as high b-carotene tomato [56,57],
canola [58], Golden Rice [59,60] and Golden Potato
[61]. Our results provide a novel example on
how subtle changes in the sequences of these pro-
teins may have drastic effects on their biosynthetic
capacities.
Experimental procedures
Strains and growth conditions
FKMC1995 is a wild-type strain of F. fujikuroi (Gibber-
ella fujikuroi mating population C) [62]. SF1 is a spontane-
ous nitrate reductase mutant obtained from FKMC1995
upon growth selection on media supplemented with KClO
3
[25]. SF4 is a carotenoid-overproducing mutant obtained
from SF1 conidia exposed to N-methyl-N¢-nitro-N-nitroso-
guanidine [63]. SF21 is a yellow-pigmented mutant obtained
by the same procedure from SF4 (Table 1). SF21 was
deposited in the Spanish Type Culture Collection (‘Cole-
ccio
´
n Espan
˜
ola de Cultivos Tipo’, University of Valencia,
Burjasot, Spain) under the name CECT 20527.
For carotenoid analysis of F. fujikuroi, strains were

grown for 9 days at 22 °C on DG minimal medium [63]
with 3 gÆ L
)1
l-asparagine instead of NaNO
3
(DGasn med-
ium). Light-grown cultures were exposed to 5 WÆm
)2
pro-
duced by a battery of five white fluorescent lights (Sylvania
Standard F36 ⁄ 154-t8).
For northern blot analyses, 4 · 10
6
conidia were grown
on 100 mL of DGasn broth in 15-cm Petri dishes for
3 days at 30 °C in the dark. For light-induction, cultures
were exposed to 25 WÆm
)2
white light for 1 h at 30 °C.
Mycelia were then immediately collected, dried, frozen in
liquid nitrogen and stored at )80 °C.
Recombinant DNA procedures
Genomic DNA was extracted according to Giordano et al.
[64] from mycelial samples harvested and washed by filtra-
tion, frozen in liquid nitrogen, and ground to a fine powder
in a cold mortar. Sequences of the carB alleles were
Alteration of Fusarium phytoene desaturase A. Prado-Cabrero et al.
4592 FEBS Journal 276 (2009) 4582–4597 ª 2009 The Authors Journal compilation ª 2009 FEBS
obtained from two overlapping PCR products generated
with primers 5¢-TGGGCGAGCTCATGAGCGACATT

AAGAAATCTG-3¢ and 5¢-CGCTCAGAACGACACCG
TTTG-3¢, covering 11 bp upstream of the start codon and
the first 957 bp of the carB coding sequence, and 5¢-
CGTTGAGGCACTGGTTAACG-3¢ and 5¢-CGAGAAT
CATGGACATAGAC-3¢, covering the last coding 1048
and 88 bp downstream of the stop codon. The sequences
of each allele were determined from two clones obtained
from independent PCR, and compared with that of the
wild-type F. fujikuroi strain FKMC1995 (accession number
AJ426418). DNA sequencing was accomplished by Newbio-
technic (Seville, Spain) using an ABI Prism
Ò
3100 Genetic
Analyser (Applied Biosystems, Foster City, CA, USA).
PCR was performed with 50 ng genomic DNA, 0.2 mm
dNTPs, 1 lm of each primer and 0.5 lL of the Expand PCR
System (Boehringer-Mannheim, Mannheim, Germany).
Reaction mixtures were heated at 94 °C for 2 min followed
by 35 cycles of denaturation (94 °C for 30 s), annealing
(55 °C for 30 s) and polymerization (68 °C for 1 min), and
by a final polymerization at 68 °C for 10 min. Amplified
DNA fragments were purified using Wizard Minicolumns
(Promega, Madison, WI, USA), and cloned into pGEM-T
Easy vector (Promega).
carB allelic replacement
Targeted mutagenesis with the SF21 carB36 allele was per-
formed following the two-step replacement method
described by Ferna
´
ndez-Martı

´
n et al. [20]. SF1 protoplasts
were prepared and transformed with the plasmid pB21H,
carrying the carB36 allele, following the protocol described
by Proctor et al. [65]. To construct pB21H, the carB allele
was amplified with primers 5¢-CAGCTTGCTCACAAT
CATCC-3¢ and 5¢-CTCATTCCAAGCCCGAGAAAC-3¢.
The 4.5-kb product was purified with the GFX
Ô
PCR DNA
and Gel Band Purification Kits GFX (Amersham Biosciences,
Piscataway, NJ, USA) and ligated to pGEM-T Easy (Pro-
mega). The carB allele was then excised using NotI and ligated
into NotI-treated pHJA2 [20], yielding plasmid pB21H.
Transformants and revertants were identified by restric-
tion analyses of PCR fragments obtained with primers Car-
BG-2F (5¢-TGGGCGAGCTCATGAGCGACATTAAGAA
ATCTG-3¢) and CarBG-3R (5¢-CGCTCAGAACGACA
CCGTTTG-3¢). The presence of the carB36 mutation was
checked using a FokI (Takara Shuzo, Kyoto, Japan) restriction
site occurring in the wild-type and absent in the mutant allele.
For Southern blot analysis, 5 lg total DNA was
digested, separated in 0.8% agarose electrophoresis, trans-
ferred to a nylon filter and hybridized with a carB probe
labelled with the nonisotopic digoxigenin labelling kit
(Roche, Mannheim, Germany) by Klenow, according to
the manufacturer. The probe was obtained from wild-type
genomic DNA by PCR with the primers CarBG-2F and
CarBG-3R.
RNA extraction and northern hybridization

For northern blot analysis, 15 lg of total RNA, extracted
with the Perfect RNA eukaryotic mini kit (Eppendorf,
Hamburg, Germany), was applied. Transferred RNA was
stained for 5 min in 0.02% methylene blue ⁄ 0.3 m sodium
acetate, pH 5.2, and rRNA bands were used as load con-
trols. Filters were hybridized with probes labelled with the
nonisotopic digoxigenin labelling kit (Roche). Nonisotop-
ically labelled single-stranded antisense probes of genes car-
RA and carB were generated by asymmetric PCR, as
described previously [66]. Initial PCR was achieved with
primers 5¢-TCCGGCGCATTTCCTATC-3¢ (forward) and
5¢-ATCTATGAATCTATGACCTC-3¢ (reverse) for gene
carRA and 5¢-GGTACTGGTGTTCCTGTCTG-3¢ (for-
ward) and 5¢-CCGATCAGATAGTTGTCACG-3¢ (reverse)
for gene carB. The resulting PCR products were used as
substrates for subsequent asymmetric amplifications with
their respective reverse primers. Signal intensities were esti-
mated by densitometric analysis using the imagej 1.42q
software ( />Plasmid constructions for carB expression in
E. coli
For the cloning of carB and carB36 cDNAs, 5 lg of total
RNA, isolated from wild-type and SF21 mycelia grown for
1 h under white light, were subjected to cDNA synthesis
using SuperScript
Ô
RNaseH
-
reverse transcriptase (Invitro-
gen, Paisley, UK) following the manufacturer’s instructions.
Two microlitres of cDNA were used for the amplification of

carB using the primers 5¢-ATGAGCGACATTAAGAA
ATCTG-3¢ and 5¢-CTAATTCGCAGCAATGACAAG-3¢.
The PCR was performed using 500 nm of each primer,
150 lm dNTPs and 1 unit of Phusion
Ô
High-Fidelity DNA
Polymerase (Finnzymes, Espoo, Finland) in the buffer
provided by the manufacturer. The reactions consisted of 30 s
of initial denaturation at 98 °C, 32 cycles of 98 °C for 15 s,
58 °C for 30 s and 72 °C for 90 s and 10 min of final polymer-
ization at 72 °C. The resulting products were purified, cloned
into the pJET1.2 ⁄ blunt
Ô
cloning vector (Fermentas, Vlinius,
Lithuania) to yield pJET–CaRB and pJET–CaRB36, respec-
tively, and confirmed by sequencing. The same amplified
cDNAs products were cloned afterwards into pBAD ⁄
THIO-TOPO
Ò
TA (Invitrogen), allowing the expression of a
thio-fusion under the control of an arabinose-inducible pro-
moter, to yield pThio–CarB and pThio–CarB36, respectively.
For c-carotene production, the al-2 cDNA was obtained
from total N. crassa RNA extracted from 2-day-old mycelial
samples grown at 30 °C in 25 mL liquid Vogel’s media. Five
micrograms of total RNA were subjected to cDNA synthesis
as described above. Two microlitres of the cDNA were then
used for amplification of al-2 using the primers 5¢-TCC
AAGCTTCTATATGACAATAGCGCC-3¢ and 5¢-CCAG
GATCCGTCTACTGCTCATACAAC-3¢, deduced from the

A. Prado-Cabrero et al. Alteration of Fusarium phytoene desaturase
FEBS Journal 276 (2009) 4582–4597 ª 2009 The Authors Journal compilation ª 2009 FEBS 4593
public sequence database (accession no. XM_960632) and
carrying a HindIII and a BamHI restriction site, respectively.
The resulting PCR product was purified and cloned into the
pCR2.1
Ò
-TOPO
Ò
vector (Invitrogen) to yield pCR–AL2.
The product was verified by sequencing. A DNA fragment
harbouring the lac promoter upstream of the al-2 cDNA was
amplified by PCR from pCR–AL2 using the primers 5¢-
AAGCGGCCGCGCGCCCAATACGC-3¢ and 5¢-CCGT
TTCTAGAGGATCCGTCTACTGC-3¢, which contain a
NotI and a XbaI restriction site, respectively. The obtained
fragment was digested with NotI and XbaI and ligated into
accordingly treated pFarbeR, which enables lycopene accu-
mulation [34], to yield pGamma, allowing the synthesis of c-
carotene upon induction with isopropyl b-d-thiogalactoside.
Validity of the constructs was verified by sequencing.
E. coli in vivo tests for CarB activity
Phytoene-, lycopene- and c-carotene-accumulating E. coli
strains were generated by transforming TOP 10 cells with
the plasmids pPhytoene [34], pFarbeR and pGamma,
respectively. Carotenoid-accumulating cells were then
transformed with pThio–CarB, pThio–CarB36 and the
control plasmid pThio (pBAD ⁄ THIO-TOPO
Ò
TA). Fifty

millilitres of LB medium containing kanamycin
(50 lgÆmL
)1
), ampicillin (100 lgÆmL
)1
) and 0.2 mm isopro-
pyl b-d-thiogalactoside, the latter to induce the expression
of the genes for the synthesis of phytoene, lycopene and
c-carotene, were grown overnight. Afterwards, the cultures
were grown at 28 °CtoD
600
= 0.5 and induced with
0.2% arabinose (w ⁄ v). Forty millilitres of the cultures were
harvested after reaching D
600
 1.2. Carotenoids were
extracted from the cell pellets by sonication in 20 mL
CHCl
3
⁄ MeOH (2 : 1, v ⁄ v). After centrifugation, epiphases
were discarded and organic phases were isolated. Interpha-
ses were then re-extracted with 20 mL CHCl
3
. The organic
phases were combined, dried under vacuum and resus-
pended in 100 lL CHCl
3
, of which 10 lL were subjected
to HPLC.
Chemical analyses

For carotenoid analysis, mycelial samples were separated
from agar and lyophilized before extraction. Carotenoids
were extracted and analysed as described by Arrach et al.
[67]. Neutral carotenoids were separated from neurospora-
xanthin by Al
2
O
3
chromatography [68]. The composition of
neutral carotenoid mixtures was determined by HPLC as
described by Arrach et al. [67]. HPLC analyses for each
strain were performed at least three times. TLC was carried
out on Silica gel 60 (Sigma) run with toluene ⁄ light petro-
leum 5 : 95 v ⁄ v.
Carotenoid analyses of E. coli cells were performed by
HPLC using a Waters system Alliance 2695 (Eschborn,
Germany) equipped with a photodiode array detector
(model 996) and a YMC-Pack C30-reversed phase column
(250 · 4.6 mm i.d., 5 lm; YMC Europe, Schermbeck,
Germany). The column was developed at a flow-rate of
1mLÆmin
)1
with a linear gradient from 100% B (MeOH ⁄
TBME ⁄ H
2
O 5:1:1 v⁄ v ⁄ v) to 50% A (MeOH ⁄ TBME
1:4 v⁄ v) within 15 min, then to 100% A within 7 min,
followed by another 10 min under the same conditions. The
flow-rate was then enhanced to 2 mLÆmin
)1

, and the sepa-
ration was continued for further 8 min. Using a Maxplot
(400–500 nm), coloured carotenoid peaks were integrated at
their individual k
max
, whereas phytoene and phytofluene
peaks were integrated at 286 and 348 nm, respectively.
Normalization and quantification were performed using an
internal a-tocopherol acetate standard according to Hoa
et al. [69]. Carotenoid amounts were calculated according
to a b-carotene standard curve. Peaks were then normalized
to correct for their individual molar extinction coefficients
[70] relative to that of b-carotene, using the correction
factors 1.970 for phytoene, 1.820 for phytofluene, 1.650 for
f-carotene, 0.806 for c-carotene, 0.720 for lycopene, 0.774
for torulene and 0.800 for 3,4-didehydrolycopene.
Sequence and secondary structure analysis
Alignments were carried out with the multi-processor ver-
sion 1.81 of the clustal w program using the server of
the Centre for Molecular and Biomolecular Informatics
(Nijmegen, The Netherlands). Secondary structure ana-
lysis was carried out with the threading program 3d-pssm
[38].
Acknowledgements
We are indebted to Dr Peter Beyer for valuable discus-
sions. We thank Erdmann Scheffer for skilful technical
assistance. This work was supported by the Spanish
Government (Ministerio de Ciencia y Tecnologı
´
a, pro-

jects BIO2003-01548 and BIO2006-01323), the Andalu-
sian Government (project P07-CVI-02813), a grant to
Dr Peter Beyer by the Bill & Melinda Gates Founda-
tion as part of the Grand Challenges in Global Health
Initiative, and the Deutsche Forschungsgemeinschaft
(DFG) Grant AL892-1-4.
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