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 o
Trang 1b-carotene biosynthesis in Fusarium fujikuroi by a point mutation in the phytoene desaturase gene
Alfonso Prado-Cabrero1, Patrick Schaub2, Violeta Dı´az-Sa´nchez1, Alejandro F Estrada1,
Salim Al-Babili2and Javier Avalos1
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.
Trang 2tion 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 C40polyene 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 accumulapigmenta-tion 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.
Trang 3end-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
cargenes 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 crassacoincides 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 reaccycliza-tion 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
Trang 4Identification 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
carBallele, 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.
ClO3K resistance
White
plasmid loss
Yellow
plasmid loss
Yellow
plasmid loss
Orange
a
carS mutations are tentatively assigned to a single hypothetical carS gene.bcarB37 and carB38 alleles include also the carB36 mutation.
Trang 5colonies 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-carc-car-otene, 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)1dry 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.
Trang 6colonies 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 quantactiv-ity 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.
Trang 7Further 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-carotphyto-ene (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
carBgene, 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], makcarotene-bind-ing 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
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.
Trang 8mutants 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
B A
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.
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.
Trang 9(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 reacdesatura-tion, 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)1dry mass.
Trang 10which 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 recogniconforma-tion 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.