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 ª 2009 The Authors Journal compilation ª 2009 FEBS tion syndromes and cardiovascular diseases [7]. In addition, carotenoids are responsible for the pigmenta- tion of some birds, insects, fish and crustaceans. Most naturally occurring carotenoids share a typical chemical structure derived from the C 40 polyene chain of the colourless precursor phytoene, a carotene syn- thesized by the enzyme phytoene synthase through the condensation of two geranylgeranyl pyrophosphate molecules (Fig. 1). Carotenoid biosynthetic pathways proceed through the sequential introduction of conju- gated double bonds in the phytoene backbone to yield increasingly desaturated molecules absorbing visible light. Desaturation steps are usually followed by cycli- zation reactions catalysed by carotene cyclases. The generated end-rings may be further modified by differ- ent oxidases introducing oxygen-containing functional groups. Carotenoids are divided into carotenes consist- ing of hydrocarbons and their oxygenated derivatives the xanthophylls [8]. Desaturation steps are achieved by a group of enzymes, with phytoene desaturases (PDSs) as their most representative members. PDS enzymes differ in the number of introduced double bonds, which range from two to five [9]. Some PDS-related enzymes desat- urate substrates other than phytoene, e.g. hydroxyneu- rosporene [10], dehydrosqualene [11] or f-carotene [12]. Plants, algae and cyanobacteria employ two enzymes, PDS and f-carotene desaturase, to perform the four desaturation reactions required for lycopene formation [13]. These enzymes are evolutionarily related to each other and to the hydroxyneurosporene dehydrogenase of Rhodobacter sphaeroides [10], but show low sequence similarity to other bacterial counterparts like the Pantoea phytoene desaturase CrtI. The low sequence conservation suggests a convergent evolution of both groups, further substantiated by their different sensitivities to chemical inhibitors [9]. Other PDS- related enzymes act as isomerases [14], e.g. the plant and cyanobacterial prolycopene isomerase CrtISO [15], or as saturases, e.g. the animal all-trans-retinol:all- trans-13,14-dihydroretinol saturase RetSat [16]. Many fungal species are useful tools for the produc- tion of secondary metabolites and the analysis of their biosyntheses. One example is the ascomycete Fusari- um fujikuroi (Gibberella fujikuroi MP-C), known for its ability to produce gibberellins [17], growth-promoting plant hormones with agricultural applications. Upon illumination, F. fujikuroi develops an orange pigmenta- tion caused by the accumulation of neurosporaxanthin [18], a carboxylic apocarotenoid originally found in the fungus Neurospora crassa [19]. Neurosporaxanthin is produced from phytoene through five desaturations, an end-cyclization, an oxidative cleavage reaction and a final oxidation step (Fig. 1). This pathway is medi- ated by the PDS CarB [20,21], the bifunctional phyto- ene synthase ⁄ carotene cyclase CarRA [21], the carotenoid cleaving oxygenase CarT [22] and finally by the presumed aldehyde dehydrogenase CarD, which is currently under investigation. F. fujikuroi also accumu- lates minor amounts of b-carotene [18] resulting from Fig. 1. Carotenoid and retinal biosynthesis in Fusarium fujikuroi. The pathway involves CarRA, CarB, the cleaving oxygenases CarX and CarT, and a postulated dehydrogenase CarD. Desaturations introduced by the CarB enzyme are circled. The grey arrow indi- cates the reaction affected in the SF21 mutant. Reactions under-represented in this strain are shaded. A. Prado-Cabrero et al. Alteration of Fusarium phytoene desaturase FEBS Journal 276 (2009) 4582–4597 ª 2009 The Authors Journal compilation ª 2009 FEBS 4583 end-cyclization of the intermediate c-carotene, cataly- sed by CarRA (Fig. 1). b-Carotene is the substrate for CarX, a second carotenoid-cleaving oxygenase, which produces retinal [23,24]. Expression of the identified car genes is stimulated by light and derepressed in the dark in carotenoid-overproducing mutants, generically called carS [22,23,25]. The mutated regulatory gene(s) responsible for the carS phenotype remains to be iden- tified. As in F. fujikuroi, a single desaturase gene has been found in other carotenogenic fungi: the ascomycetes N. crassa (al-1) [26] and Cercospora nicotianae (pdh1) [27], the zygomycetes Phycomyces blakesleeanus, Mu- cor circinelloides and Blakeslea trispora (carB) [28–30] and the basidiomycete Xhanthophyllomyces dendror- hous (crtI) [31], formerly Phaffia rhodozyma. These enzymes, more similar to those of carotenogenic bacte- ria than to desaturases of photosynthetic organisms, are presumably responsible for all desaturation steps in the corresponding carotenoid pathways. The ability to carry out four desaturations was first inferred from genetic approaches for the CarB PDS from P. blakes- leeanus [32], and later confirmed by heterologous expression in Escherichia coli [28]. A similar heterolo- gous approach demonstrated the ability of CrtI from X. dendrorhous to catalyse the four desaturations from phytoene to lycopene [31] and of AL-1 from N. crassa to achieve the five desaturations from phytoene to 3,4- didehydrolycopene [33]. The carotenoid pathway of N. crassa coincides with that of F. fujikuroi in the syn- thesis of the same end-product, the apocarotenoid neurosporaxanthin, but both fungi differ in the order of the reactions. Whereas in N. crassa the five desatu- rations are performed first and followed by cyclization reaction as a later step [34], in F. fujikuroi the cycliza- tion reaction precedes the fourth and fifth desatura- tion steps, as indicated by the absence of lycopene and the occurrence of b-zeacarotene in different strains [18]. The accumulation of phytoene in carB mutants [20] and the lack of strains blocked in a later desaturation step indicated that CarB is responsible for all five desaturations. Here, we provide conclusive evidence for the ability of the CarB enzyme to carry out the five desaturation reactions and to discriminate between different carotenoid substrates. We have isolated and characterized a F. fujikuroi carB mutant impaired in the catalysis of the fifth desaturation (i.e. that con- verting c-carotene to torulene), but fully able to cata- lyse the preceding four desaturation steps. The effect of the mutation was confirmed by targeted allele replacement and comparing the activity of wild-type and altered CarB enzymes in different carotenoid- producing E. coli strains. Finally, a hypothesis is proposed to explain the structural basis of the effect of the mutation. Results Isolation and phenotypic analysis of a yellow mutant The pale pigmentation of wild-type F. fujikuroi hinders the identification of colour mutants with alterations in the carotenoid pattern. Such mutants are easily identi- fied in deeply orange-pigmented strains like the carS carotenoid-overproducing mutants [18]. A screening for colour mutants was performed after chemical mutagenesis of the carS strain SF4, a descendent of the nitrate reductase-deficient mutant SF1 (Table 1), not affected in carotenoid biosynthesis and formerly used as a recipient strain for transformation experi- ments [25]. This search led to the identification of a mutant with a striking yellow colour (Fig. 2). This mutant was subcultured from single conidia and denominated as SF21. The carotenoids produced by SF21 were analysed by spectrophotometry, TLC and HPLC (Fig. 2). As indicated by their colours, UV ⁄ Vis spectra of the SF21 carotenoid samples differed from that of its ancestor strain SF4 in shape and maximal absorption. TLC analyses revealed that most of the carotenoids accumulated by SF4 were highly polar, pointing to neurosporaxanthin as the predominant component. The minor neutral fraction contained torulene and traces of other carotene intermediates. Parallel separa- tion of the SF21 crude carotenoid samples revealed two predominant bands corresponding to c-carotene and b-carotene. In contrast to SF4, no torulene could be detected, and the neurosporaxanthin band was much paler. HPLC analyses of the neutral carotenoid fractions from both strains confirmed the predomi- nance of torulene in SF4 and the accumulation of large amounts of c-carotene and b-carotene in SF21 (Fig. 2). The amount of phytoene was low in both strains, but was significantly higher in SF21 than in SF4. Quantification of the carotenoid contents in mycelial samples from light- or dark-grown cultures showed similar results, except for a higher neurosporaxanthin content in the illuminated SF4 samples (Fig. 2). As expected, its parental strain SF1 produced moderate amounts of neurosporaxanthin only in the light. The carotenoid concentration was at least threefold higher in SF21 than in SF4, with neurosporaxanthin repre- senting < 10% of the total carotene. Alteration of Fusarium phytoene desaturase A. Prado-Cabrero et al. 4584 FEBS Journal 276 (2009) 4582–4597 ª 2009 The Authors Journal compilation ª 2009 FEBS Identification of a mutation in the SF21 carB allele The carotenoid pattern of the mutant SF21, i.e. the accumulation of c-carotene and the subsequent deviation of the pathway to b-carotene, suggested impaired c-carotene to torulene desaturation activity. This may be the result of an altered CarB if all five desaturations required for torulene synthesis are catalysed by this sole F. fujikuroi PDS enzyme (Fig. 1). To test this hypothesis, we cloned and sequenced the carB alleles from strains SF21 and wild-type FKMC1995. The carB gene was formerly cloned from the wild- type F. fujikuroi strain IMI58289 [21] and its sequence was deposited in the EMBL database (accession num- ber AJ426418). The carB sequence from FKMC1995 was identical to that of IMI58289 except for a C196 fi T transition which does not affect the encoded protein sequence. The predicted CarB protein shared a similar structural organization with other PDS and PDS-related enzymes of different origins (Fig. 3), including the characteristic N-terminal dinu- cleotide-binding domain [35,36]. Compared with carB from FKMC1995, the SF21 carB allele, designated here as carB36, showed a single point mutation, a C608 fi T transition, resulting in a Pro170 fi Leu substitution. The corresponding resi- due is located in a predicted a-helix-rich region (Fig. 3) far from the presumed carotene binding domain harbouring the mutations formerly identified in three P. blakesleaanus carB mutants [37]. Replacement of the wild-type carB allele by carB36 The generation of mutant SF21 from wild-type FKMC1995 includes two chemical mutagenesis steps (Table 1), presumably resulting in further random mutations in addition to that found in the SF21 carB36 allele. To check if this allele is sufficient to pro- duce the deviation of the pathway to b-carotene in a wild-type carotenogenesis background, a two-step strategy was used to replace the carB allele of strain SF1 with carB36 (Fig. 4A,C). Ten transformants were isolated after transformation of the SF1 strain with a plasmid carrying carB36. In five of them, Southern blot analyses showed the incorporation of a single copy of the plasmid at the carB locus (Fig. 4A,B). Three of these strains were checked for carotenoid content. Compared with the wild-type, the three trans- formants contained approximately twofold more carot- enoids upon illumination, but exhibited similar carotenoid compositions. One of these transformants (T5, indicated by an asterisk in Fig. 4B) was chosen for further investigation. T5 conidia were grown on Petri dishes to search for mutant colonies, expected at low frequency from spontaneous plasmid loss by homologous recombination (Fig. 4C). Transfer of indi- vidual colonies to selective medium showed variable frequencies of hygromycin sensitive strains, usually > 1%. However, all the strains tested were orange and contained the wild-type carB allele, suggesting preferential recombination through the same DNA segment that led to the plasmid integration. No yellow Table 1. Fusarium fujikuroi strains used in this study. Only the relevant transformant is included. For clarity, wild-type carB alleles (carB + ) are also indicated. NG, N-methyl-N¢-nitro-N-nitrosoguanidine. Strain Genotype a,b Origin Colour in the dark FKMC1995 carB + White SF1 niaD4 carB + FKMC1995, spontaneous ClO 3 K resistance White SF4 niaD4 carS35 carB + SF1, NG mutagenesis Orange SF21 niaD4 carS35 carB36 SF4, NG mutagenesis Yellow SF73 niaD4 carS35 carB37 SF21, NG mutagenesis Greenish SF98 niaD4 carS35 carB38 SF21, NG mutagenesis Pale greenish T5 niaD4 carB + ⁄ carB36 hygR SF1, transformation with pB21H White SF191 niaD4 carS63 carB + ⁄ carB36 hygR T5, NG mutagenesis Orange SF214 niaD4 carS63 carB36 SF191, spontaneous plasmid loss Yellow SF215 niaD4 carS63 carB36 SF191, spontaneous plasmid loss Yellow SF216 niaD4 carS63 carB + SF191, spontaneous plasmid loss Orange a carS mutations are tentatively assigned to a single hypothetical carS gene. b carB37 and carB38 alleles include also the carB36 mutation. A. Prado-Cabrero et al. Alteration of Fusarium phytoene desaturase FEBS Journal 276 (2009) 4582–4597 ª 2009 The Authors Journal compilation ª 2009 FEBS 4585 colonies were detected after visual inspection of at least 120 Petri dishes with  250–500 colonies, proba- bly because of the difficult identification of this pheno- type in the pale pigmented background of T5. Because SF21 was obtained from a carotenoid-over- producing strain, a mutagenesis experiment was used to obtain a T5-derived carS mutant, termed here SF191. This deregulated strain contained more carote- noids than SF4 (Fig. 5A), as expected from the pres- ence of two carB genes, one with the carB36 mutation (Fig. 4A). Hygromycin-sensitive strains were obtained from SF191 and checked by PCR for the loss of the carB36 allele. One of them, called SF216, harboured a single wild-type carB allele (PCR test not shown) and had a lower carotenoid content (Fig. 5A) than SF191. In contrast to SF4, SF216 contained similar amounts of carotenoids in dark or light, indicating differences in their respective carS mutations. Conidia collected from SF191 were grown in the same media and screened for the generation of yellow Fig. 2. SF21 phenotype. Representative colonies of SF4 and SF21 strains grown in the dark at 22 °C on DGasn agar. TLC and HPLC analy- ses of carotenoid samples from 9-day-old mycelia of both strains grown under the same conditions. UV ⁄ Vis spectra (350–550 nm) and maxi- mal absorbance wavelengths (nm) of accumulated carotenoids are shown in the insets. Below: quantitative analyses of the carotenoids produced by SF1, SF4 and SF21. A scheme of the pathway is presented on the left. Phytoene, phytofluene, f-carotene, b-zeacarotene, c-car- otene, b-carotene, torulene and neurosporaxanthin are abbreviated as P, Pf, f, b-z, c, b, T and Nx, respectively. The identities of the interme- diates are depicted by colour. Surfaces are proportional to amounts, indicated in lgÆg )1 dry mass. The data show average and standard deviation (outer semicircles) from three independent determinations. Left and right semicircles correspond to cultures grown in the dark and under continuous light, respectively. SF1 contained only trace amounts of carotenoids in the dark. SF4 contained low amounts of phytoene, c-carotene and b-carotene, represented as approximate calculations. Circles missing in the SF4 and SF21 schemes correspond to undetected carotenoids. Alteration of Fusarium phytoene desaturase A. Prado-Cabrero et al. 4586 FEBS Journal 276 (2009) 4582–4597 ª 2009 The Authors Journal compilation ª 2009 FEBS colonies. Two yellow colonies were identified in a screening of  5000; both strains, called SF214 and SF215, were sensitive to hygromycin, indicating loss of the integrated plasmid. As predicted, both mutants lacked the FokI restriction site, present in the wild-type carB allele, but not in the carB36 mutant allele (Fig. 4D), confirming the expected allele replacement. Like SF21, SF214 and SF215 contained low amounts of neurosporaxanthin. Moreover, HPLC analyses of their neutral carotenoid fractions showed a pattern very similar to that of SF21 either in dark- or in light- grown cultures (Fig. 5B). This result strongly indicated that the carB36 mutation is responsible for the yellow phenotype, i.e. the defective CarB capacity to carry out the fifth desaturation step in the neurosporaxan- thin biosynthetic pathway. Heterologous expression of the carB36 allele To further confirm the effect of the carB36 mutation on enzyme activity, wild-type carB and carB36 cDNAs were cloned and expressed in different carotene-pro- ducing E. coli strains and the resulting carotene patterns were determined (Fig. 6). Expression of wild- type carB in a phytoene producing E. coli strain resulted in an efficient desaturation to lycopene accom- panied by a lower production of 3,4-didehydrolycopene, indicating that the fifth desaturation is less efficiently achieved than the preceding four in the E. coli back- ground. Expression of carB36 resulted in lycopene amounts at least comparable with those formed by CarB, whereas the production of 3-4 didehydrolyco- pene was reduced approximately sixfold (Fig. 6A). Similar results were obtained through introducing the two cDNAs in a lycopene-producing E. coli strain, expressing the bacterial four-step desaturase gene crtI. As shown in Fig. 6B, the activities of CarB and CarB36 led to similar lycopene contents, whereas the amounts of 3,4-didehydrolycopene were approximately eightfold higher in the carB-expressing strain. The carotenoid pattern of F. fujikuroi indicates that the substrate for the fifth desaturation step is c-caro- tene rather than lycopene. Therefore, we expressed the two cDNAs in a c-carotene-accumulating E. coli strain, engineered by introduction of the bacterial desaturase CrtI and the N. crassa cyclase ⁄ phytoene synthase AL-2 [34]. Compared with CarB36, the activ- ity of CarB led to a sevenfold higher quantity of toru- lene (Fig. 6C). Similarly, the conversion of lycopene to 3,4-didehydrolycopene, achieved in parallel in the same cells, was much higher in CarB-expressing cells. Taken together, the usage of the E. coli system confirmed the specific effect of the carB36 mutation on the fifth desaturation reaction. Fig. 3. Alignment of predicted structures for phytoene desaturases from the fungi Fusarium fujikuroi (CarB Ff, accession number CAD19989.2), Neurospora crassa (AL-1 Nc, XP964713), Xhanthophyllomyces dendrorhous (CrtI Xd, AAO53257) and Phycomyces blakeslee- anus (CarB Pb, CAA55197.1), the bacteria Rhodobacter sphaeroides (CrtI Rs, YP353345), the archaea Sulfolobus solfataricus (CrtI Ss, NP344226), and the plant Arabidopsis thaliana (PDS3 At, Q07356). The comparison also includes the A. thaliana f-carotene desaturase (ZDS1 At, Q38893) and the human all-trans-retinol 13,14-reductase (RetSat Hs, Q6NUM9). Structures were deduced with the program 3D-PSSM. Broad rectangles represent predicted a helices, and thin rectangles represent predicted b sheets. The conserved b–a–b dinucleo- tide-binding domain is indicated in black. The a-helix-rich segment is shaded in grey. Polarity of the helices of this region and the presence of basic residues in the F. fujikuroi enzyme are indicated ( • ,hydrophilic; , moderately hydrophobic; s, highly hydrophobic; each short line below is either a lysine or an arginine residue). Vertical lines and arrows indicate mutations; SF21, Pro170 fi Leu; SF73, Trp449 fi Stop; SF98, Gly504 fi Asp (described in this work); A486, Glu426 fi Lys; C5, Ser444 fi Phe and Leu446 fi Phe; S442, Glu482 fi Lys, described by Sanz et al. [37]. The asterisk marks the mutation in the R. sphaeroides PDS enzyme that provides the ability to carry out a fourth desaturation. A. Prado-Cabrero et al. Alteration of Fusarium phytoene desaturase FEBS Journal 276 (2009) 4582–4597 ª 2009 The Authors Journal compilation ª 2009 FEBS 4587 Further alterations in carB activity To determine further residues essential for other desat- uration steps, mutagenesis experiments of the SF21 strain were performed, leading to the isolation of low- pigmented strains. Two of them were SF73 and SF98, which exhibited a pale greenish hue. HPLC analyses of these two mutants revealed the accumulation of phyto- ene and lower amounts of f-carotene (Fig. 7). SF98 also contained minor amounts of c-carotene and b-car- otene, whereas SF73 was hardly able to desaturate f-carotene. These SF73 and SF98 carotene patterns suggested the occurrence of further mutations in the carB gene, resulting in more impaired PDSs. Sequence analysis of the corresponding carB genes showed a G1493 fi A transition in the SF73 carB allele, resulting in a Trp449 fi Stop mutation. The predicted truncated protein lacks the C-terminal 121 amino acids, which include the putative carotene-bind- ing domain [37], making the accumulation of minor amounts of carotenoids an unexpected result. The SF98 carB allele contains a G1657 fi A transition, leading to a Gly504 fi Asp replacement in the caro- tene-binding domain (Fig. 3). The phenotype of the SF73 and SF98 mutants could be also caused by a combined effect of these mutations with the carB36 Pro170 fi Leu substitution, also present in these strains. Relation between carotenoid biosynthesis and expression of the car genes in carS and carS ⁄ carB mutants As shown in Figs 7 and 8, the striking difference in the carotenoid content between SF21 and its precursor strain SF4 was less pronounced in the SF21-derived A BD C Fig. 4. carB allele replacement. (A) Physical map of the pB21H integration at the homologous carB sequence by a single recombination event in the genome of strain SF1. The mutation in the carB36 allele is indicated by a star. The carB probe, relevant BamHI sites used for Southern blot analyses and expected fragment sizes are indicated. (B) Southern blot analyses of the recipient strain SF1 and 10 transfor- mants. Squares highlight transformants whose hybridization pattern indicates the incorporation of a single copy of the plasmid at the carB locus. The transformant T5 was used in further experimental steps. (C) Physical map of molecular events leading to loss of the plasmid pB21H by a single recombination at the homologous carB sequence in the genome of a carS strain derived from T5. The recombination shown occurs at the opposite side from the one that produced the plasmid integration, leaving the mutated carB allele in the genome. (D) Electrophoretic profiles of the PCR products obtained with primers flanking the mutation site at allele carB36 using DNA from wild-type, SF21, SF191, SF214 and SF215 strains and digested with FokI. Interpretation of expected bands is depicted on the right scheme. The SF21 mutation leads to the loss of a FokI restriction site. Alteration of Fusarium phytoene desaturase A. Prado-Cabrero et al. 4588 FEBS Journal 276 (2009) 4582–4597 ª 2009 The Authors Journal compilation ª 2009 FEBS mutants SF73 and SF98. Furthermore, the latter mutants regained the light induction of carotenogene- sis, which had disappeared in the parental strain SF21 (Fig. 8). To check whether the differences in carotene content are a result of altered mRNA levels for the carotenogenic enzymes, we carried out northern blot experiments. As expected, carRA or carB mRNAs were undetect- able in dark-grown mycelia of the wild-type and SF1 strains and highly induced after 1 h exposure to light BA Fig. 5. Effect of wild-type and carB36 alleles on Fusarium fujikuroi carotenogenesis. (A) Carotenoids accumulated by the mutants SF1, SF4, SF21, SF191 and SF216. (B) Neutral carotenoids accumulated by the mutants SF21, SF214 and SF215. The analyses were carried out on mycelial samples from dark or light-grown cultures (5 WÆm )2 ). The data show average of two independent experiments. ABC Fig. 6. CarB36 desaturation activity of CarB and CarB36 in Escherichia coli strains producing different carotene substrates. Based on HPLC analyses, the data show carotenoid compositions of three E. coli strains accumulating different carotenoid intermediates and expressing wild-type thioredoxin-carB,-carB36 or thioredoxin (control). The three E. coli strains were engineered by introducing the following enzymes: (A) phytoene synthase (phytoene accumulation in the control); (B) phytoene synthase and the bacterial desaturase CrtI (lycopene accumula- tion); (C) phytoene synthase, CrtI and the Neurospora phytoene synthase ⁄ lycopene cyclase AL-2 (lycopene and c-carotene accumulation). The data show average and standard deviation of three independent experiments. 3,4ddl = 3,4-didehydrolycopene. A. Prado-Cabrero et al. Alteration of Fusarium phytoene desaturase FEBS Journal 276 (2009) 4582–4597 ª 2009 The Authors Journal compilation ª 2009 FEBS 4589 (Fig. 8). mRNA levels in dark-grown SF4 were similar to those of illuminated wild-type and SF1 strains and exhibited a significant increase because of light expo- sure, correlating with enhanced carotenoid production. Similar expression patterns were observed for SF21, SF73 and SF98, indicating that the SF21 increased carotenoid content is not caused by enhanced tran- script levels. Discussion The carotenoid biosynthetic pathway of the orange- pigmented F. fujikuroi includes a sequence of five desaturation steps, consisting of two pairs of equivalent reactions at symmetrical sites in the carotene skeleton, predictably interrupted by a cyclization of the interme- diate neurosporene and completed by a fifth reaction in an outer position to produce torulene (Fig. 1). We have identified a yellow-pigmented mutant, SF21, exhibiting a novel carotenoid pattern. Consistent with its deep yellow colour, SF21 accumulates large amounts of c- and b-carotene and minor amounts of the final product neurosporaxanthin, indicating a specific defect on the CarB ability to catalyse the fifth desaturation reaction. Previous studies indicated that CarB is responsible for all desaturation steps of the pathway. Lack of mutants whose end product is any of the partially desaturated intermediates was inter- preted as an indication of the achievement of the five reactions by a single desaturase [18]. The investigations of the carB36 allele and the encoded enzyme, reported here, provide solid support to this assumption. More- over, our results show that CarB36 desaturase is unique in the specific impairment of the fifth desatura- tion reaction, which is caused by a single amino acid exchange in the wild-type enzyme. CarB shares a similar structural organization with other PDS enzymes, as revealed by our secondary structure predictions using the 3d-pssm protein-fold recognition program [38]. The same overall structure, including the b–a–b dinucleotide-binding domain [35,36], is displayed by phylogenetically distant PDS- related enzymes like the f-carotene desaturase from Arabidopsis thaliana, which shows only 14% sequence identity to CarB. Several carB mutations formerly investigated in the zygomycete P. blakesleeanus are located in a region close to the carboxy-end of the pro- tein [37], tentatively associated with binding of the carotenoid substrate [39]. One of these mutants, S442, exhibits a defective PDS with partial activity for the first two desaturations, leading to the accumulation of significant amounts of f-carotene [40]. In this study, we identified two pale greenish strains, the SF21- derived mutants SF73 and SF98, exhibiting a pheno- type similar to that of the P. blakesleeanus S442 caused by mutations affecting the same protein domain. The different carotenoid patterns of SF73 and SF98 reflect defective PDSs maintaining certain activities with respect to the first pair of reactions but different capacities to perform the second pair of desaturations. The leaky activity of the SF98 desaturase resulted in the accumulation of significant amounts of f-carotene and c-carotene. However, we could not detect their respective precursors in the pathway, i.e. phytofluene or b-zeacarotene, indicating that when a desaturation reaction occurs, the symmetric reaction is readily achieved. A similar result was found with the mutant SF73, possessing a more severely impaired desaturase, Fig. 7. Scheme of the carotenoids accumulated by mutants SF21, SF73 and SF98 under continuous illumination. The pathway on the left includes only detected carotenoids. Phytoene, f-carotene, c-carotene, b-carotene, and neurosporaxanthin are abbreviated as P, f, c, b and Nx, respectively. Circle surfaces are proportional to carotenoid amounts, indicated in lgÆg )1 dry mass. Alteration of Fusarium phytoene desaturase A. Prado-Cabrero et al. 4590 FEBS Journal 276 (2009) 4582–4597 ª 2009 The Authors Journal compilation ª 2009 FEBS which lost the ability to carry out the second pair of desaturations but maintained a low, but significant, capacity to produce f-carotene. However, despite its low desaturating activity, no phytofluene was accumu- lated. Interestingly, the SF73 desaturase represents a truncated CarB lacking the C-terminus, which includes the presumed substrate-binding domain. Hence, partic- ipation of other protein segments in carotene binding must be concluded. The carB36 mutation is located in a predicted a-helix-rich protein domain, apparently distant from the carboxy domain formerly interpreted as involved in carotene binding. A single mutation in the same a- helix-rich domain of the three-step PDS of Rhodobacter sphaeroides (Fig. 3) allows this enzyme to recognize neurosporene as a substrate [41], supporting a relevant role for this PDS segment in substrate recognition. This a-helix-rich domain is similar to the proposed membrane surface-binding domain of protoporphyri- nogen IX oxidase (PPO) [42], an enzyme structurally related to PDSs. The PPO domain is characterized by the presence of amphipathic a helices rich in basic amino acids that interact with the phospholipid head groups of the lipid bilayer, embedding partially into the membrane and constituting a pore, which enables entrance of the hydrophobic substrate. As in other organisms, fungal PDSs are membrane-bound proteins [43,44] that act on hydrophobic substrates occurring in the lipid bilayer. The a helices of the PDS domain mentioned above have different hydrophobicities, and five of them contain basic amino acids that could interact with phospholipid head groups (Fig. 3). PDS enzymes might employ a membrane-binding and sub- strate-uptake mechanism similar to that of PPO. The Fusarium carB36 mutation could alter the conforma- tion of a putative pore, preventing the recognition and ⁄ or entrance of c-carotene. The proline residue replaced in the predicted F. fu- jikuroi CarB36 protein is found in the PDSs AL-1 from N. crassa and CrtI from X. dendrorhous, presum- ably able to carry out five desaturations [33,45,46]. Conversely, the PDSs from the b-carotene-producing zygomycetes M. circinelloides, P. blakesleeanus and B. trispora, which carry out only four desaturations, contain an aliphatic residue instead of proline at the same position. However, this rule seems not to be valid for the PDS of the ascomycete C. nicotiane, described as producing b-carotene [27], because this enzyme is highly similar to CarB from F. fujikuroi ( 70% identical amino acids), including the con- served proline residue. Carotene analysis of the close relative C. cruenta shows different carotenoids, but none of them result from a fifth desaturation [47]. Based on our observations, a side branch of the carotenoid pathway in C. nicotiane involving a fifth desaturation cannot be discarded. This is actually the case in X. dendrorhous, where the four-desaturation pathway into b-carotene and astaxanthin coexists with a lateral production of torulene [45,46]. The preva- lence of astaxanthin biosynthesis implies a highly effi- cient cyclase activity, which competes with the fifth desaturation step. Former studies proposed a mechanism of action for fungal PDSs organized as oligomers. In P. blakeslee- Fig. 8. Total carotenoid contents and mRNA levels for genes carRA and carB in the wild-type and the mutants SF1, SF4, SF21, SF73 and SF98. D: grown in the dark. L: grown under continuous illumi- nation. Northern blot analyses were performed with total RNA sam- ples. rRNA bands are shown below each panel as load controls. The bars below each northern blot show the ratios of signal intensi- ties to rRNA controls; the values are expressed relative to the maximum in each panel, taken as 1. A. Prado-Cabrero et al. Alteration of Fusarium phytoene desaturase FEBS Journal 276 (2009) 4582–4597 ª 2009 The Authors Journal compilation ª 2009 FEBS 4591 [...]... enzyme, CarB36 could then be impaired in the ability to incorporate c-carotene rather than in the desaturase activity The total amount of carotenoids accumulated by SF21 is at least three times higher than in its neurosporaxanthin- overproducing precursor strain SF4, although their carRA and carB mRNA levels remain unaltered The amount of carotenoids in vivo is presumably determined by the balance between... two overlapping PCR products generated with primers 5¢-TGGGCGAGCTCATGAGCGACATT AAGAAATCTG-3¢ and 5¢-CGCTCAGAACGACACCG TTTG-3¢, covering 11 bp upstream of the start codon and the first 957 bp of the carB coding sequence, and 5¢CGTTGAGGCACTGGTTAACG-3¢ and 5¢-CGAGAAT CATGGACATAGAC-3¢, covering the last coding 1048 and 88 bp downstream of the stop codon The sequences of each allele were determined from... Estrada AF, Al-Babili S & Avalos J (2007) Identification and biochemical characterization of a novel carotenoid oxygenase: elucidation of the cleavage step in the Fusarium carotenoid pathway Mol Microbiol 64, 448–460 23 Thewes S, Prado-Cabrero A, Prado MM, Tudzynski B & Avalos J (2005) Characterization of a gene in the car cluster of Fusarium fujikuroi that codes for a protein of the carotenoid oxygenase... (5¢-TGGGCGAGCTCATGAGCGACATTAAGAA ATCTG-3¢) and CarBG-3R (5¢-CGCTCAGAACGACA CCGTTTG-3¢) The presence of the carB36 mutation was checked using a FokI (Takara Shuzo, Kyoto, Japan) restriction site occurring in the wild-type and absent in the mutant allele For Southern blot analysis, 5 lg total DNA was digested, separated in 0.8% agarose electrophoresis, 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) in the buffer provided by the 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 by a higher activity or the CarB36 enzyme compared with the wild-type counterpart or by a higher stability of c-carotene and b-carotene compared with torulene and neurosporaxanthin This may actually be the case for torulene, as indicated by the fast decoloration of a torulene-accumulating... For example, replacement of the carB wild-type allele by carB36 in Fusarium venenatum, a fungus used by the food industry as the source for mycoprotein [50], may result in the predominant synthesis of c-carotene and b-carotene and give added value to mycoprotein for human consumption Furthermore, a similar mutation in 4592 X dendrorhous could deviate the biosynthesis 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 of Fusarium phytoene desaturase A 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 of Fusarium phytoene desaturase A 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