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
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Deviationoftheneurosporaxanthinpathway towards
b-carotene biosynthesisinFusariumfujikuroibya point
mutation inthephytoenedesaturase 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 Fusariumfujikuroi (Gibberella fujikuroi mating population C) develops
an orange pigmentation caused by an accumulation ofthe carboxylic apoc-
arotenoid neurosporaxanthin. The synthesis of this xanthophyll includes
five desaturation steps presumed to be catalysed bythe carB-encoded phy-
toene desaturase. In this study, we identified a yellow mutant (SF21) by
mutagenesis ofa carotenoid-overproducing strain. HPLC analyses indi-
cated a specific impairment inthe ability of SF21-CarB to perform the fifth
desaturation, as implied bythe accumulation of c-carotene and b-carotene,
which arise through four-step desaturation. Sequencing ofthe SF21 carB
allele revealed a single mutation resulting in an exchange ofa residue con-
served in other five-step desaturases. Targeted carB allele replacement
proved that this single mutation is the cause ofthe 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 ofthe 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 inthe 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 bythe 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 inthephytoene 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 bya 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 phytoenedesaturase 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 bythe 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 bythe 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 ofb-carotene [18] resulting from
Fig. 1. Carotenoid and retinal biosynthesis
in Fusarium fujikuroi. Thepathway involves
CarRA, CarB, the cleaving oxygenases CarX
and CarT, and a postulated dehydrogenase
CarD. Desaturations introduced bythe CarB
enzyme are circled. The grey arrow indi-
cates the reaction affected inthe SF21
mutant. Reactions under-represented in this
strain are shaded.
A. Prado-Cabrero et al. Alteration ofFusariumphytoene desaturase
FEBS Journal 276 (2009) 4582–4597 ª 2009 The Authors Journal compilation ª 2009 FEBS 4583
end-cyclization ofthe 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 ofthe 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 desaturasegene 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. fujikuroiinthe syn-
thesis ofthe same end-product, the apocarotenoid
neurosporaxanthin, but both fungi differ inthe 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. fujikuroithe cycliza-
tion reaction precedes the fourth and fifth desatura-
tion steps, as indicated bythe absence of lycopene
and the occurrence of b-zeacarotene in different
strains [18].
The accumulation ofphytoenein carB mutants [20]
and the lack of strains blocked ina later desaturation
step indicated that CarB is responsible for all five
desaturations. Here, we provide conclusive evidence for
the ability ofthe 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 ofthe 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 themutation 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 ofthe effect
of the mutation.
Results
Isolation and phenotypic analysis ofa 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 ofthe 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 ofthe 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 ofthe 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 theneurosporaxanthin band was
much paler. HPLC analyses ofthe 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-carotenein SF21
(Fig. 2). The amount ofphytoene was low in both
strains, but was significantly higher in SF21 than in
SF4.
Quantification ofthe carotenoid contents in mycelial
samples from light- or dark-grown cultures showed
similar results, except for a higher neurosporaxanthin
content inthe illuminated SF4 samples (Fig. 2). As
expected, its parental strain SF1 produced moderate
amounts ofneurosporaxanthin only inthe light. The
carotenoid concentration was at least threefold higher
in SF21 than in SF4, with neurosporaxanthin repre-
senting < 10% ofthe total carotene.
Alteration ofFusariumphytoenedesaturase A. Prado-Cabrero et al.
4584 FEBS Journal 276 (2009) 4582–4597 ª 2009 The Authors Journal compilation ª 2009 FEBS
Identification ofamutationinthe SF21 carB
allele
The carotenoid pattern ofthe mutant SF21, i.e. the
accumulation of c-carotene and the subsequent
deviation ofthepathway 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 inthe 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 ina 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 ofthe 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 inthe SF21
carB36 allele. To check if this allele is sufficient to pro-
duce thedeviationofthepathway to b-carotenein 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 ofthe SF1 strain with a
plasmid carrying carB36. In five of them, Southern
blot analyses showed the incorporation ofa single
copy ofthe 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. Fusariumfujikuroi 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 inthe 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 ofFusariumphytoene 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 ofthe difficult identification of this pheno-
type inthe 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 inthe 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 inthe insets. Below: quantitative analyses ofthe carotenoids
produced by SF1, SF4 and SF21. A scheme ofthepathway 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 ofthe 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 inthe dark and
under continuous light, respectively. SF1 contained only trace amounts of carotenoids inthe dark. SF4 contained low amounts of phytoene,
c-carotene and b-carotene, represented as approximate calculations. Circles missing inthe SF4 and SF21 schemes correspond to undetected
carotenoids.
Alteration ofFusariumphytoenedesaturase 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 inthe wild-type
carB allele, but not inthe 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 inthe neurosporaxan-
thin biosynthetic pathway.
Heterologous expression ofthe carB36 allele
To further confirm the effect ofthe 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 inaphytoene producing E. coli strain
resulted in an efficient desaturation to lycopene accom-
panied bya lower production of 3,4-didehydrolycopene,
indicating that the fifth desaturation is less efficiently
achieved than the preceding four inthe 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 ina lycopene-producing E. coli strain,
expressing the bacterial four-step desaturasegene 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 inthe 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 ina c-carotene-accumulating E. coli
strain, engineered by introduction ofthe 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 inthe same
cells, was much higher in CarB-expressing cells. Taken
together, the usage ofthe E. coli system confirmed the
specific effect ofthe carB36 mutation on the fifth
desaturation reaction.
Fig. 3. Alignment of predicted structures for phytoene desaturases from the fungi Fusariumfujikuroi (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 ofthe helices of this region and the presence
of basic residues inthe 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 themutationinthe R. sphaeroides PDS enzyme that provides the ability to carry out a
fourth desaturation.
A. Prado-Cabrero et al. Alteration ofFusariumphytoene 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 ofthe 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 ofthe corresponding carB genes
showed a G1493 fi A transition inthe SF73 carB
allele, resulting ina 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 inthe 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 ofthe 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 inthe SF21-derived
A
BD
C
Fig. 4. carB allele replacement. (A) Physical map ofthe pB21H integration at the homologous carB sequence bya single recombination
event inthe genome of strain SF1. Themutationinthe carB36 allele is indicated bya star. The carB probe, relevant BamHI sites used for
Southern blot analyses and expected fragment sizes are indicated. (B) Southern blot analyses ofthe recipient strain SF1 and 10 transfor-
mants. Squares highlight transformants whose hybridization pattern indicates the incorporation ofa single copy ofthe plasmid at the carB
locus. The transformant T5 was used in further experimental steps. (C) Physical map of molecular events leading to loss ofthe plasmid
pB21H bya single recombination at the homologous carB sequence inthe genome ofa 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 inthe genome. (D)
Electrophoretic profiles ofthe PCR products obtained with primers flanking themutation 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 ofa FokI restriction site.
Alteration ofFusariumphytoenedesaturase 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 inthe 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 ofthe 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 Fusariumfujikuroi carotenogenesis. (A) Carotenoids accumulated bythe mutants SF1, SF4,
SF21, SF191 and SF216. (B) Neutral carotenoids accumulated bythe 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 inthe 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 deviationof three independent experiments. 3,4ddl = 3,4-didehydrolycopene.
A. Prado-Cabrero et al. Alteration ofFusariumphytoene 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 pathwayofthe orange-
pigmented F. fujikuroi includes a sequence of five
desaturation steps, consisting of two pairs of equivalent
reactions at symmetrical sites inthe carotene skeleton,
predictably interrupted bya cyclization ofthe interme-
diate neurosporene and completed bya 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 ofthe achievement ofthe five
reactions bya 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 inthe specific impairment ofthe fifth desatura-
tion reaction, which is caused bya single amino acid
exchange inthe 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 inthe zygomycete P. blakesleeanus are
located ina region close to the carboxy-end ofthe 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 ofthe 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 ofthe SF98 desaturase resulted in
the accumulation of significant amounts of f-carotene
and c-carotene. However, we could not detect their
respective precursors inthe 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 ofthe carotenoids accumulated by mutants SF21, SF73 and SF98 under continuous illumination. Thepathway 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 ofFusariumphytoenedesaturase 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 ina predicted
a-helix-rich protein domain, apparently distant from
the carboxy domain formerly interpreted as involved
in carotene binding. A single mutationinthe same a-
helix-rich domain ofthe 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 ofthe lipid bilayer, embedding partially into
the membrane and constituting a pore, which enables
entrance ofthe hydrophobic substrate. As in other
organisms, fungal PDSs are membrane-bound proteins
[43,44] that act on hydrophobic substrates occurring in
the lipid bilayer. Thea helices ofthe 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 ofa putative pore, preventing the recognition
and ⁄ or entrance of c-carotene.
The proline residue replaced inthe predicted F. fu-
jikuroi CarB36 protein is found inthe 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 ofthe 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 ofthe 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 pathwayin 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 inthe wild-type and the mutants SF1, SF4, SF21, SF73
and SF98. D: grown inthe 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 ofFusariumphytoene desaturase
FEBS Journal 276 (2009) 4582–4597 ª 2009 The Authors Journal compilation ª 2009 FEBS 4591
[...]... enzyme, CarB36 could then be impaired inthe ability to incorporate c-carotene rather than inthedesaturase activity The total amount of carotenoids accumulated by SF21 is at least three times higher than in its neurosporaxanthin- overproducing precursor strain SF4, although their carRA and carB mRNA levels remain unaltered The amount of carotenoids in vivo is presumably determined bythe balance between... two overlapping PCR products generated with primers 5¢-TGGGCGAGCTCATGAGCGACATT AAGAAATCTG-3¢ and 5¢-CGCTCAGAACGACACCG TTTG-3¢, covering 11 bp upstream ofthe start codon and the first 957 bp ofthe carB coding sequence, and 5¢CGTTGAGGCACTGGTTAACG-3¢ and 5¢-CGAGAAT CATGGACATAGAC-3¢, covering the last coding 1048 and 88 bp downstream ofthe stop codon The sequences of each allele were determined from... Estrada AF, Al-Babili S & Avalos J (2007) Identification and biochemical characterization ofa novel carotenoid oxygenase: elucidation ofthe cleavage step intheFusarium carotenoid pathway Mol Microbiol 64, 448–460 23 Thewes S, Prado-Cabrero A, Prado MM, Tudzynski B & Avalos J (2005) Characterization ofageneinthe car cluster ofFusariumfujikuroi that codes for a protein ofthe carotenoid oxygenase... (5¢-TGGGCGAGCTCATGAGCGACATTAAGAA ATCTG-3¢) and CarBG-3R (5¢-CGCTCAGAACGACA CCGTTTG-3¢) The presence ofthe carB36 mutation was checked using a FokI (Takara Shuzo, Kyoto, Japan) restriction site occurring inthe wild-type and absent inthe mutant allele For Southern blot analysis, 5 lg total DNA was digested, separated in 0.8% agarose electrophoresis, transferred to a nylon filter and hybridized with a carB... 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 PhusionÔ High-Fidelity DNA Polymerase (Finnzymes, Espoo, Finland) inthe buffer provided bythe manufacturer The reactions consisted of 30 s of initial denaturation at 98 °C, 32 cycles of 98... biosynthetic activities and carotenoid degradation rates Therefore, the higher carotenoid content in SF21 may be explained either bya higher activity or the CarB36 enzyme compared with the wild-type counterpart or bya higher stability of c-carotene and b-carotene compared with torulene and neurosporaxanthin This may actually be the case for torulene, as indicated bythe fast decoloration ofa torulene-accumulating... For example, replacement ofthe carB wild-type allele by carB36 inFusarium venenatum, a fungus used bythe food industry as the source for mycoprotein [50], may result inthe predominant synthesis of c-carotene and b-carotene and give added value to mycoprotein for human consumption Furthermore, a similar mutationin 4592 X dendrorhous could deviate thebiosynthesis further toward b-carotene and, therefore,... al-2 using the primers 5¢-TCC AAGCTTCTATATGACAATAGCGCC-3¢ and 5¢-CCAG GATCCGTCTACTGCTCATACAAC-3¢, deduced from the FEBS Journal 276 (2009) 4582–4597 ª 2009 The Authors Journal compilation ª 2009 FEBS 4593 Alteration ofFusariumphytoenedesaturaseA Prado-Cabrero et al public sequence database (accession no XM_960632) and carrying a HindIII and a BamHI restriction site, respectively The resulting PCR... under the same conditions The flow-rate was then enhanced to 2 mLÆmin)1, and the separation was continued for further 8 min Using a Maxplot (400–500 nm), coloured carotenoid peaks were integrated at their individual kmax, 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...Alteration ofFusariumphytoenedesaturaseA Prado-Cabrero et al anus, each PDS monomer carries out a single desaturation and transfers the desaturated product to the next monomer, the process being repeated up to the accomplishment 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 incorporate partially desaturated . 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 ,. 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 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