The ylo-1 gene encodes an aldehyde dehydrogenase responsible for the last reaction in the Neurospora carotenoid pathway

The accumulation of the apocarotenoid neurosporaxanthin and its carotene precursors explains the orange pigmentation of the Neurospora surface cultures. Neurosporaxanthin biosynthesis requires the activity of the albino gene products (AL-1, AL-2 and AL-3), which yield the precursor torulene. Recently, we identified the carotenoid oxygenase CAO-2, which cleaves torulene to produce the aldehyde β-apo-4'-carotenal. This revealed a last missing step in Neurospora carotenogenesis, namely the oxidation of the CAO-2 product to the corresponding acid neurosporaxanthin. The mutant ylo-1 , which exhibits a yellow colour, lacks neurosporaxanthin and accumulates several carotenes, but its biochemical basis is unknown. Based on available genetic data, we identified ylo-1 in the Neurospora genome, which encodes an enzyme representing a novel subfamily of aldehyde dehydrogenases, and demonstrated that it is responsible for the yellow phenotype, by sequencing and complementation of mutant alleles. In contrast to the precedent structural genes in the carotenoid pathway, light does not induce the synthesis of ylo-1 mRNA. In vitro incubation of purified YLO-1 protein with β-apo-4'-carotenal produced neurosporaxanthin through the oxidation of the terminal aldehyde into a carboxyl group. We conclude that YLO-1 completes the set of enzymes needed for the synthesis of this major Neurospora pigment.     

The Arabidopsis thaliana REDUCED EPIDERMAL FLUORESCENCE1 gene encodes an aldehyde dehydrogenase involved in ferulic acid and sinapic acid biosynthesis

Recent research has significantly advanced our understanding of the phenylpropanoid pathway but has left in doubt the pathway by which sinapic acid is synthesized in plants. The reduced epidermal fluorescence1 (ref1) mutant of Arabidopsis thaliana accumulates only 10 to 30% of the sinapate esters found in wild-type plants. Positional cloning of the REF1 gene revealed that it encodes an aldehyde dehydrogenase, a member of a large class of NADP(+)-dependent enzymes that catalyze the oxidation of aldehydes to their corresponding carboxylic acids. Consistent with this finding, extracts of ref1 leaves exhibit low sinapaldehyde dehydrogenase activity. These data indicate that REF1 encodes a sinapaldehyde dehydrogenase required for sinapic acid and sinapate ester biosynthesis. When expressed in Escherichia coli, REF1 was found to exhibit both sinapaldehyde and coniferaldehyde dehydrogenase activity, and further phenotypic analysis of ref1 mutant plants showed that they contain less cell wall-esterified ferulic acid. These findings suggest that both ferulic acid and sinapic acid are derived, at least in part, through oxidation of coniferaldehyde and sinapaldehyde. This route is directly opposite to the traditional representation of phenylpropanoid metabolism in which hydroxycinnamic acids are instead precursors of their corresponding aldehydes.     

The P450-4 gene of Gibberella fujikuroi encodes ent-kaurene oxidase in the gibberellin biosynthesis pathway

At least five genes of the gibberellin (GA) biosynthesis pathway are clustered on chromosome 4 of Gibberella fujikuroi; these genes encode the bifunctional ent-copalyl diphosphate synthase/ent-kaurene synthase, a GA-specific geranylgeranyl diphosphate synthase, and three cytochrome P450 monooxygenases. We now describe a fourth cytochrome P450 monooxygenase gene (P450-4). Gas chromatography-mass spectrometry analysis of extracts of mycelia and culture fluid of a P450-4 knockout mutant identified ent-kaurene as the only intermediate of the GA pathway. Incubations with radiolabeled precursors showed that the metabolism of ent-kaurene, ent-kaurenol, and ent-kaurenal was blocked in the transformants, whereas ent-kaurenoic acid was metabolized efficiently to GA(4). The GA-deficient mutant strain SG139, which lacks the 30-kb GA biosynthesis gene cluster, converted ent-kaurene to ent-kaurenoic acid after transformation with P450-4. The B1-41a mutant, described as blocked between ent-kaurenal and ent-kaurenoic acid, was fully complemented by P450-4. There is a single nucleotide difference between the sequence of the B1-41a and wild-type P450-4 alleles at the 3' consensus sequence of intron 2 in the mutant, resulting in reduced levels of active protein due to a splicing defect in the mutant. These data suggest that P450-4 encodes a multifunctional ent-kaurene oxidase catalyzing all three oxidation steps between ent-kaurene and ent-kaurenoic acid.     

A carotenoid biosynthesis gene cluster in Fusarium fujikuroi: the genes carB and carRA

Phytoene synthase, phytoene dehydrogenase and carotene cyclase are three of the four enzyme activities needed to produce the acidic carotenoid neurosporaxanthin from the precursor geranylgeranyl pyrophosphate. In the filamentous fungus Fusarium fujikuroi, these three enzyme activities are encoded by two closely linked genes, carRA and carB, oriented in the same direction in the genome. The two genes are separated by 548 bp and code for two polypeptides of 612 and 541 amino acids, respectively, which are highly similar to the homologous proteins from other filamentous fungi. The ORF of carRA contains a 96-bp insertion that is absent in the other fungal homologues. The 32 additional residues are located in one of the two repeated domains responsible for the cyclase activity in the homologous fungal proteins. We have determined the function of carRA by gene disruption. The resulting mutants were albino and had lost the ability to produce phytoene, as expected from the simultaneous loss of phytoene synthase and carotene cyclase. In the same experiments, we also found transformants in which carB had been deleted. These mutants accumulate phytoene, confirming the function of the gene previously shown by gene-targeted mutagenesis. Expression of carRA and carB is strongly induced by light. Loss of carB or disruption of the carRA ORF led to enhanced expression of the carRA gene, suggesting the existence of a feedback regulatory mechanism.     

Segregation of secondary metabolite biosynthesis in hybrids of Fusarium fujikuroi and Fusarium proliferatum

Fusarium fujikuroi and Fusarium proliferatum are two phylogenetically closely related species of the Gibberella fujikuroi species complex (GFC). In some cases, strains of these species can cross and produce a few ascospores. In this study, we analyzed 26 single ascospore isolates of an interspecific cross between F. fujikuroi C1995 and F. proliferatum D4854 for their ability to produce four secondary metabolites: gibberellins (GAs), the mycotoxins fusarin C and fumonisin B(1), and a family of red polyketides, the fusarubins. Both parental strains contain the biosynthetic genes for all four metabolites, but differ in their ability to produce these metabolites under certain conditions. F. fujikuroi C1995 produces GAs and fusarins, while F. proliferatum D4854 produces fumonisins and fusarubins. The segregation amongst the progeny of these traits is not the expected 1:1 Mendelian ratio. Only eight, six, three and three progeny, respectively, produce GAs, fusarins, fumonisin B(1) and fusarubins in amounts similar to those synthesized by the producing parental strain. Beside the eight highly GA(3)-producing progeny, some of the progeny produce small amounts of GAs, predominantly GA(1), although these strains contain the GA gene cluster of the non-GA-producing F. proliferatum parental strain. Some progeny had recombinant secondary metabolite profiles under the conditions examined indicating that interspecific crosses can yield secondary metabolite production profiles that are atypical of the parent species.     

Linkage among genes responsible for fumonisin biosynthesis in Gibberella fujikuroi mating population A.

Most naturally occurring strains of the fungus Gibberella fujikuroi mating population A produce high levels of the mycotoxin fumonisin B1 (FB1), which is oxygenated at both carbons C-5 and C-10. Some strains, however, produce only FB2 or FB3, suggesting that they lack the ability to hydroxylate position C-10 or C-5, respectively. Genetic analysis indicates that these different phenotypes are due to single gene defects at closely linked loci designated fum2 and fum3. Further allellism tests indicate that both fum2 and fum3 are closely linked to fum1, a previously identified gene that regulates fumonisin production. The recovery frequency of FB1-producing progency from cross 510 between fum1 and fum2 mutations suggests a map distance of approximately 6.2 cM between these two loci. Amplified fragment length polymorphism analysis of parents and progeny of cross 510 was employed to confirm that the FB1-producing strains are recombinant progeny. We conclude that fum1, fum2, and fum3 constitute a fumonisin biosynthetic gene cluster on chromosome 1 of the restriction fragment length-map of G. fujikuroi.     

Retinal biosynthesis in fungi: characterization of the carotenoid oxygenase CarX from Fusarium fujikuroi

The car gene cluster of the ascomycete Fusarium fujikuroi encodes two enzymes responsible for torulene biosynthesis (CarRA and CarB), an opsin-like protein (CarO), and a putative carotenoid cleaving enzyme (CarX). It was presumed that CarX catalyzes the formation of the major carotenoid in F. fujikuroi, neurosporaxanthin, a cleavage product of torulene. However, targeted deletion of carX did not impede neurosporaxanthin biosynthesis. On the contrary, DeltacarX mutants showed a significant increase in the total carotenoid content, indicating an involvement of CarX in the regulation of the pathway. In this work, we investigated the enzymatic activity of CarX. The expression of the enzyme in beta-carotene-accumulating Escherichia coli cells led to the formation of the opsin chromophore retinal. The identity of the product was proven by high-performance liquid chromatography and gas chromatography-mass spectrometry. Subsequent in vitro assays with heterologously expressed and purified CarX confirmed its beta-carotene-cleaving activity and revealed its capability to produce retinal also from other substrates, such as gamma-carotene, torulene, and beta-apo-8'-carotenal. Our data indicate that the occurrence of at least one beta-ionone ring in the substrate is required for the cleavage reaction and that the cleavage site is determined by the distance to the beta-ionone ring. CarX represents the first retinal-synthesizing enzyme reported in the fungal kingdom so far. It seems likely that the formed retinal is involved in the regulation of the carotenoid biosynthetic pathway via a negative feedback mechanism.     

Fusarium Tri4 encodes a multifunctional oxygenase required for trichothecene biosynthesis

Fusarium graminearum and Fusarium sporotrichioides produce the trichothecene mycotoxins 15-acetyldeoxynivalenol and T-2 toxin, respectively. In both species, disruption of the P450 monooxygenase-encoding gene, Tri4, blocks production of the mycotoxins and leads to the accumulation of the trichothecene precursor trichodiene. To further characterize its function, the F. graminearum Tri4 (FgTri4) was heterologously expressed in the trichothecene-nonproducing species Fusarium verticillioides. Transgenic F. verticillioides carrying the FgTri4 converted exogenous trichodiene to the trichothecene biosynthetic intermediates isotrichodermin and trichothecene. Conversion of trichodiene to isotrichodermin requires seven biochemical steps. The fifth and sixth steps can occur nonenzymatically. Precursor feeding studies done in the current study indicate that wild-type F. verticillioides has the enzymatic activity necessary to carry out the seventh step, the C-3 acetylation of isotrichodermol to form isotrichodermin. Together, the results of this study indicate that the Tri4 protein catalyzes the remaining four steps and is therefore a multifunctional monooxygenase required for trichothecene biosynthesis.     

The NADPH-cytochrome P450 reductase gene from Gibberella fujikuroi is essential for gibberellin biosynthesis

The fungus Gibberella fujikuroi is used for the commercial production of gibberellins (GAs), which it produces in very large quantities. Four of the seven GA biosynthetic genes in this species encode cytochrome P450 monooxygenases, which function in association with NADPH-cytochrome P450 reductases (CPRs) that mediate the transfer of electrons from NADPH to the P450 monooxygenases. Only one cpr gene (cpr-Gf) was found in G. fujikuroi and cloned by a PCR approach. The encoded protein contains the conserved CPR functional domains, including the FAD, FMN, and NADPH binding motifs. cpr-Gf disruption mutants were viable but showed a reduced growth rate. Furthermore, disruption resulted in total loss of GA(3), GA(4), and GA(7) production, but low levels of non-hydroxylated C(20)-GAs (GA(15) and GA(24)) were still detected. In addition, the knock-out mutants were much more sensitive to benzoate than the wild type due to loss of activity of another P450 monooxygenase, the detoxifying enzyme, benzoate p-hydroxylase. The UV-induced mutant of G. fujikuroi, SG138, which was shown to be blocked at most of the GA biosynthetic steps catalyzed by P450 monooxygenases, displayed the same phenotype. Sequence analysis of the mutant cpr allele in SG138 revealed a nonsense mutation at amino acid position 627. The mutant was complemented with the cpr-Gf and the Aspergillus niger cprA genes, both genes fully restoring the ability to produce GAs. Northern blot analysis revealed co-regulated expression of the cpr-Gf gene and the GA biosynthetic genes P450-1, P450-2, P450-4 under GA production conditions (nitrogen starvation). In addition, expression of cpr-Gf is induced by benzoate. These results indicate that CPR-Gf is the main but not the only electron donor for several P450 monooxygenases from primary and secondary metabolism.     

The structural gene for the mitochondrial aldehyde dehydrogenase maps to human chromosome 12

Summary A cloned 850 bp cDNA fragment corresponding to the 3-coding part of human ALDHI-mRNA was used as a probe for the chromosomal assignment of the ALDHI gene. Southern blot analysis of human-rodent somatic cell hybrids indicates that the human ALDHI gene resides on chromosome 12.Dedicated to Prof. Dr. H. Holzer on the occasion of his 65. birthday     

The Pseudomonas oleovorans alkBAC operon encodes two structurally related rubredoxins and an aldehyde dehydrogenase

The Pseudomonas oleovorans alkBAC operon encodes seven proteins, of which at least three are involved in alkane hydroxylase (alkBA) and alkanol dehydrogenase (alkC) activities. We have determined the nucleotide sequence of the 2.5-kilobase pair alkA region and analyzed the role of its translation products in alkane oxidation. The alkA region contains three coding sequences, encoding two related rubredoxins (alkF and alkG) of 14- and 18-kDa molecular mass and a 52-kDa aldehyde dehydrogenase (alkH). Deletion analysis indicated that neither the 14-kDa alkF gene product (rubredoxin 1) nor the amino-terminal part of the 18-kDa alkG gene product (rubredoxin 2) is required for alkane hydroxylase activity in vivo. The product of the alkH cistron restores growth of a P. oleovorans aldehyde dehydrogenase mutant on aliphatic alcohols and aldehydes. Its amino acid sequence shows considerable homology to previously characterized aldehyde dehydrogenases from mammalian and fungal origin. The nucleotide composition of the alk genes (47% G + C) differs considerably from the G + C content of the P. oleovorans genome suggesting that the alk regulon may originate from an unrelated organism.     

Genomic structure of the human mitochondrial aldehyde dehydrogenase gene

We have isolated and characterized four overlapping clones from two cosmid human genomic libraries, which span about 90 kilobase pairs (kbp) and contain the entire human mitochondrial aldehyde dehydrogenase (ALDH2) gene. Restriction maps of the genomic clones were elucidated utilizing cDNA probes and specific oligonucleotide probes. The organization of exons and introns was established by DNA sequencing of each exon and splicing junctions. The ALDH2 gene is about 44 kbp in length and contains at least 13 exons which encode 517 amino acid residues. Except for the signal NH2-terminal peptide, which is absent in the mature enzyme, the amino acid sequence deduced from the exons coincided with the reported primary structure of human liver ALDH2 (J. Hempel, R. Kaiser, and H. J?rnvall, 1985, Eur. J. Biochem. 153: 13-28). Several introns contain Alu repetitive sequences. A TATA-like sequence (TTATAAAA) and a CAAT-like sequence (GTCATCAT) are located 473 and 515 bp, respectively, upstream from the translation initiation codon. Primer extension and S1 nuclease mapping were performed to characterize the 5'-region of the gene.     

The biosynthesis of kaurenolide diterpenoids by gibberella fujikuroi

The biosynthesis of 7β-hydroxy- and 7β,18-dihydroxy-kaurenolides from ent-kaur-16-en-19-oic acid has been investigated by incubating unlabelled