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.     

Identification of the gene responsible for torulene cleavage in the Neurospora carotenoid pathway

Torulene, a C₄₀ carotene, is the precursor of the end product of the Neurospora carotenoid pathway, the C₃₅ xanthophyll neurosporaxanthin. Torulene is synthesized by the enzymes AL-2 and AL-1 from the precursor geranylgeranyl diphosphate and then cleaved by an unknown enzyme into the C₃₅ apocarotenoid. In general, carotenoid cleavage reactions are catalyzed by carotenoid oxygenases. Using protein data bases, we identified two putative carotenoid oxygenases in Neurospora, named here CAO-1 and CAO-2. A search for novel mutants of the carotenoid pathway in this fungus allowed the identification of two torulene-accumulating strains, lacking neurosporaxanthin. Sequencing of the cao-2 gene in these strains revealed severe mutations, pointing to a role of CAO-2 in torulene cleavage. This was further supported by the identical phenotype found upon targeted disruption of cao-2. The biological function was confirmed by in vitro assays using the purified enzyme, which cleaved torulene to produce β-apo-4′-carotenal, the corresponding aldehyde of neurosporaxanthin. The specificity of CAO-2 was shown by the lack of γ-carotene-cleaving activity in vitro. As predicted for a structural gene of the carotenoid pathway, cao-2 mRNA was induced by light in a WC-1 and WC-2 dependent manner. Our data demonstrate that CAO-2 is the enzyme responsible for the oxidative cleavage of torulene in the neurosporaxanthin biosynthetic pathway.     

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.     

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.     

Investigation of factors influencing production of the monocyclic carotenoid torulene in metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Factors influencing production of the monocyclic carotenoid torulene in recombinant Escherichia coli were investigated by modulating enzyme expression level, culture conditions, and engineering of the isoprenoid precursor pathway. The gene dosage of in vitro evolved lycopene cyclase crtY2 significantly changed the carotenoid profile. A culture temperature of 28°C showed better production of torulene than 37°C while initial culture pH had no significant effect on torulene production. Glucose-containing LB, 2×YT, TB and MR media significantly repressed the production of torulene, and the other carotenoids lycopene, tetradehydrolycopene, and -carotene, in E. coli. In contrast, glycerol-containing LB, 2×YT, TB, and MR media enhanced torulene production. Overexpression of dxs, dxr, idi and/or ispA, individually and combinatorially, enhanced torulene production up to 3.1–3.3 fold. High torulene production was observed in a high dissolved oxygen level bioreactor in TB and MR media containing glycerol. Lycopene was efficiently converted into torulene during aerobic cultures, indicating that the engineered torulene synthesis pathway is well coordinated, and maintains the functionality and integrity of the carotenogenic enzyme complex.     

Novel apocarotenoid intermediates in Neurospora crassa mutants imply a new biosynthetic reaction sequence leading to neurosporaxanthin formation

Neurosporaxanthin, beta-apo-4'-carotenoic acid (C35), represents the end-product of the carotenoid pathway in Neurospora crassa. It is supposed to be synthesized in three steps catalyzed by sequential AL-2, CAO-2 and YLO-1 activities: (i) cyclization of 3,4-didehydrolycopene (C40); (ii) cleavage of torulene into beta-apo-4'-carotenal (C35); and finally (iii) oxidation of beta-apo-4'-carotenal. However, analyses of the ylo-1 mutant revealed the accumulation of intermediates other than beta-apo-4'-carotenal. Here, we generated a 3,4-didehydrolycopene accumulating Escherichia coli strain and showed that CAO-2 cleaves this acyclic carotene in vivo and in vitro yielding apo-4'-lycopenal. The apocarotenoids accumulated in the ylo-1 mutant were then identified as apo-4'-lycopenal and apo-4'-lycopenol, pointing to the former as the YLO-1 substrate and indicating that cyclization is the last step in neurosporaxanthin biosynthesis. This was further substantiated by analyses of a cyclase-deficient al-2 mutant, revealing the accumulation of apo-4'-lycopenoic acid. The three acyclic apocarotenoids presented here have not been found naturally before.     

Studies on the near-UV effect on carotenogenesis in Verticillium agaricinum

Carotenoids identified in Verticillium agaricinum under near-UV were beta-, zeta-, and gamma-carotenes, neurosporene, torulene, neurosporaxanthin and one of its esters. Evidence supports the proposal that gamma-carotene, and not torulene, is the immediate precursor of neurosporaxanthin. It is also suggested that phytochrome may be involved in the high irradiance reactions (HIR) causing carotenoid synthesis in this fungus although there is no knowledge of how this is effected. Spores grown under near-UV conditions varied in size and shape from those grown in the dark. A new pigment (390, 420 nm) is also proposed as the photoreceptor for carotenogenesis in V. agaricinum.     

Mutants of the carotene cyclase domain of al-2 from Neurospora crassa

Phytoene synthase and carotene cyclase, two key enzymes in carotenoid biosynthesis, are encoded by two separate genes in bacteria and plants, but by a single bifunctional gene in fungi. The cyclase function has been demonstrated for the products of the genes crtYB from the basidiomycete Xanthophyllomyces dendrorhous, and for carRA and carRP from the zygomycetes Phycomyces blakesleeanus and Mucor circinelloides, respectively. These three genes are highly similar to al-2 from Neurospora crassa. Taking advantage of the high proportion of the final product of the carotenoid pathway that accumulates Neurospora when mycelium is illuminated at low temperature, we have isolated two mutants with a pale reddish pigmentation. Both mutants are complemented by the wild-type al-2 gene, and carry mutations in the al-2 domain to which cyclase activity has been attributed in other fungi. The mutants lack neurosporaxanthin and accumulate an unidentified reddish carotenoid, as shown by column chromatography and HPLC. The chemical and spectrophotometrical properties of this carotenoid are consistent with the absence of carotenoid cyclization, and indicate that the product of al-2 is bifunctional. The existence of a single gene responsible for phytoene synthase and carotene cyclase thus seems to be a widespread trait among filamentous fungi, as shown by the examples now known in a basidiomycete, two zygomycetes and one ascomycete.     

The gene carD encodes the aldehyde dehydrogenase responsible for neurosporaxanthin biosynthesis in Fusarium fujikuroi

Neurosporaxanthin (β-apo-4'-carotenoic acid) biosynthesis has been studied in detail in the fungus Fusarium fujikuroi. The genes and enzymes for this biosynthetic pathway are known until the last enzymatic step, the oxidation of the aldehyde group of its precursor, β-apo-4'-carotenal. On the basis of sequence homology to Neurospora crassa YLO-1, which mediates the formation of apo-4'-lycopenoic acid from the corresponding aldehyde substrate, we cloned the carD gene of F. fujikuroi and investigated the activity of the encoded enzyme. In vitro assays performed with heterologously expressed protein showed the formation of neurosporaxanthin and other apocarotenoid acids from the corresponding apocarotenals. To confirm this function in vivo, we generated an Escherichia coli strain producing β-apo-4'-carotenal, which was converted into neurosporaxanthin upon expression of carD. Moreover, the carD function was substantiated by its targeted disruption in a F. fujikuroi carotenoid-overproducing strain, which resulted in the loss of neurosporaxanthin and the accumulation of β-apo-4'-carotenal, its derivative β-apo-4'-carotenol, and minor amounts of other carotenoids. Intermediates accumulated in the ΔcarD mutant suggest that the reactions leading to neurosporaxanthin in Neurospora and Fusarium are different in their order. In contrast to ylo-1 in N. crassa, carD mRNA content is enhanced by light, but to a lesser extent than other enzymatic genes of the F. fujikuroi carotenoid pathway. Furthermore, carD mRNA levels were higher in carotenoid-overproducing mutants, supporting a functional role for CarD in F. fujikuroi carotenogenesis. With the genetic and biochemical characterization of CarD, the whole neurosporaxanthin biosynthetic pathway of F. fujikuroi has been established.     

Metabolic engineering of the carotenoid biosynthetic pathway in the yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma)

The crtYB locus was used as an integrative platform for the construction of specific carotenoid biosynthetic mutants in the astaxanthin-producing yeast Xanthophyllomyces dendrorhous. The crtYB gene of X. dendrorhous, encoding a chimeric carotenoid biosynthetic enzyme, could be inactivated by both single and double crossover events, resulting in non-carotenoid-producing transformants. In addition, the crtYB gene, linked to either its homologous or a glyceraldehyde-3-phosphate dehydrogenase promoter, was overexpressed in the wild type and a beta-carotene-accumulating mutant of X. dendrorhous. In several transformants containing multiple copies of the crtYB gene, the total carotenoid content was higher than in the control strain. This increase was mainly due to an increase of the beta-carotene and echinone content, whereas the total content of astaxanthin was unaffected or even lower. Overexpression of the phytoene synthase-encoding gene (crtI) had a large impact on the ratio between mono- and bicyclic carotenoids. Furthermore, we showed that in metabolic engineered X. dendrorhous strains, the competition between the enzymes phytoene desaturase and lycopene cyclase for lycopene governs the metabolic flux either via beta-carotene to astaxanthin or via 3,4-didehydrolycopene to 3-hydroxy-3'-4'-didehydro-beta-psi-caroten-4-one (HDCO). The monocylic carotenoid torulene and HDCO, normally produced as minority carotenoids, were the main carotenoids produced in these strains.     

A chromoplast-specific carotenoid biosynthesis pathway is revealed by cloning of the tomato white-flower locus

Carotenoids and their oxygenated derivatives xanthophylls play essential roles in the pigmentation of flowers and fruits. Wild-type tomato (Solanum lycopersicum) flowers are intensely yellow due to accumulation of the xanthophylls neoxanthin and violaxanthin. To study the regulation of xanthophyll biosynthesis, we analyzed the mutant white-flower (wf). It was found that the recessive wf phenotype is caused by mutations in a flower-specific beta-ring carotene hyroxylase gene (CrtR-b2). Two deletions and one exon-skipping mutation in different CrtR-b2 wf alleles abolish carotenoid biosynthesis in flowers but not leaves, where the homologous CrtR-b1 is constitutively expressed. A second beta-carotene hydroxylase enzyme as well as flower- and fruit-specific geranylgeranyl diphosphate synthase, phytoene synthase, and lycopene beta-cyclase together define a carotenoid biosynthesis pathway active in chromoplasts only, underscoring the crucial role of gene duplication in specialized plant metabolic pathways. We hypothesize that this pathway in tomato was initially selected during evolution to enhance flower coloration and only later recruited to enhance fruit pigmentation. The elimination of beta-carotene hydroxylation in wf petals results in an 80% reduction in total carotenoid concentration, possibly caused by the inability of petals to store high concentrations of carotenoids other than xanthophylls and by degradation of beta-carotene, which accumulates as a result of the wf mutation but is not due to altered expression of genes in the biosynthetic pathway.     

Amplification of HMG-CoA reductase production enhances carotenoid accumulation in Neurospora crassa

Neurospora crassa, a filamentous fungus, naturally produces the carotenoids lycopene and neurosporaxanthin. To increase the carbon flux through the carotenoid biosynthetic pathway, the 1658-bp region of the HMG1 gene encoding the catalytic domain (cHMG1) of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase of Saccharomyces cerevisiae was expressed in N. crassa under control of the strong, constitutive glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter and the inducible alcohol dehydrogenase (alcA) promoter. Overexpressing cHMG1 under control of the GPD promoter increased lycopene and neurosporaxanthin production 6- and 1.5-fold, respectively, relative to the wild-type strain. Over-expression of cHMG1 under control of the alcA promoter increased production of lycopene and neurosporaxanthin 3- and 2-fold, respectively.     

A C35 carotenoid biosynthetic pathway

Upon coexpression with Erwinia geranylgeranyldiphosphate (GGDP) synthase in Escherichia coli, C(30) carotenoid synthase CrtM from Staphylococcus aureus produces novel carotenoids with the asymmetrical C(35) backbone. The products of condensation of farnesyldiphosphate and GDP, C(35) structures comprise 40 to 60% of total carotenoid accumulated. Carotene desaturases and carotene cyclases from C(40) or C(30) pathways accepted and converted the C(35) substrate, thus creating a C(35) carotenoid biosynthetic pathway in E. coli. Directed evolution to modulate desaturase step number, together with combinatorial expression of the desaturase variants with lycopene cyclases, allowed us to produce at least 10 compounds not previously described. This result highlights the plastic and expansible nature of carotenoid pathways and illustrates how combinatorial biosynthesis coupled with directed evolution can rapidly access diverse chemical structures.     

Functional analysis of beta- and epsilon-ring carotenoid hydroxylases in Arabidopsis

Lutein and zeaxanthin are dihydroxy xanthophylls that are produced from their corresponding carotene precursors by the action of beta- and epsilon -ring carotenoid hydroxylases. Two genes that encode beta-ring hydroxylases (beta-hydroxylases 1 and 2) have been identified in the Arabidopsis genome and are highly active toward beta-rings but only weakly active toward epsilon -rings. A third distinct activity required for epsilon -ring hydroxylation has been defined by mutation of the LUTEIN1 (LUT1) locus, but LUT1 has not yet been cloned. To address the individual and overlapping functions of the three Arabidopsis carotenoid hydroxylase activities in vivo, T-DNA knockout mutants corresponding to beta-hydroxylases 1 and 2 (b1 and b2, respectively) were isolated and all possible hydroxylase mutant combinations were generated. beta-Hydroxylase single mutants do not exhibit obvious growth defects and have limited impact on carotenoid composition relative to the wild type, suggesting that the encoded proteins have a significant degree of functional redundancy in vivo. Surprisingly, the b1 b2 double mutant, which lacks both known beta-hydroxylase enzymes, still contains significant levels of beta-carotene-derived xanthophylls, suggesting that additional beta-ring hydroxylation activity exists in vivo. The phenotype of double and triple hydroxylase mutants indicates that at least a portion of this activity resides in the LUT1 gene product. Despite the severe reduction of beta-carotene-derived xanthophylls (up to 90% in the lut1 b1 b2 triple mutant), the double and triple hydroxylase mutants still contain at least 50% of the wild-type amount of hydroxylated beta-rings. This finding suggests that it is the presence of minimal amounts of hydroxylated beta-rings, rather than minimal amounts of specific beta-carotene-derived xanthophylls, that are essential for light-harvesting complex II assembly and function in vivo. The carotenoid profiles in wild-type seeds and the effect of single and multiple hydroxylase mutations are distinct from those in photosynthetic tissues, indicating that the activities of each gene product differ in the two tissues. Overall, the hydroxylase mutants provide insight into the unexpected overlapping activity of carotenoid hydroxylases in vivo.     

Effect of certain nitrogenous heterocyclic compounds on carotenogenesis in Verticillium agaricinum.

Pyridine, isonicotinoylhydrazide and 1-methylamidazole have been used to investigate carotenoid biosynthesis in V. agaricinum. The results suggest that both torulin (C40) and neurosporaxanthin (C35) are formed from the precursors phytoene and phytofluene. These was no evidence of lycopene accumulation under these conditions. After 4 days' growth in the presence of isocotinolyhydrazine the fungus contained torulin and neurosporaxanthin only, whereas after 7 days, seven other carotenoids appeared as well, some of which were at the early stages of carotenoid biosynthesis. There results cannot be explained on the basis of a system consisting of free enzymes but of an enzyme aggregate already proposed for Phycomyces.     

The biotechnological potential of the al-2 gene from Neurospora [correction of Neurospra] crassa for the production of monocyclic keto hydroxy carotenoids

The al-2 cDNA from Neurospora crassa was cloned, expressed and functionally characterized. The enzyme comprised the two catalytic activities of a phytoene synthase and a lycopene cyclase. In contrast to most other lycopene cyclases, single cyclizations were preferentially catalyzed. This N. crassa enzyme is the first CrtYB-type monocyclic-acting lycopene cyclase. Therefore, this cDNA has been evaluated for the heterologous synthesis of monocyclic hydroxy-keto carotenoids by combination with other carotenogenic genes in Escherichia coli. Depending on the degree of desaturation, 4-keto derivatives of gamma-carotene and torulene with additional 2-hydroxy, 3-hydroxy and/or 1'-HO groups were generated and the following asymmetrical carotenoids identified and quantitated: 3-HO-4-keto-gamma-carotene, 2-HO-4-keto-gamma-carotene, 4-keto-1'-HO-gamma-carotene, 3,1'-(HO)(2)-4-keto-gamma-carotene, 3-HO-4-keto-torulene and 2-HO-4-keto-torulene. Among them all the monocyclic gamma-carotene derivatives with 9 conjugated double bonds were not found naturally before. Furthermore, 2-HO-4-keto-torulene with 10 conjugated double bonds is another novel carotenoid.