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: avalos@us.es
(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 ª2009 The Authors Journal compilation ª2009 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 ª2009 The Authors Journal compilation ª2009 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 ª2009 The Authors Journal compilation ª2009 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 fiT 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 fiT transition, resulting in a
Pro170 fiLeu 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 ª2009 The Authors Journal compilation ª2009 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 ª2009 The Authors Journal compilation ª2009 FEBS
