Light induction of the carotenoid biosynthesis pathway in Blakeslea trispora

A gene of Blakeslea trispora has been cloned by heterologous hybridization with the Mucor circinelloides crgA gene, a repressor of light-inducible carotenogenesis. This gene is the ortholog of the M. circinelloides crgA, since it was able to restore the wild-type phenotype of a null crgA mutant of M. circinelloides. The expression of B. trispora crgA gene is light-induced and photoadapted, as occurs for M. circinelloides crgA. Light induction and photoadaptation of B. trispora crgA was also observed in M. circinelloides, which suggests that the mechanisms involved in light regulation are basically conserved between these filamentous fungi. Conservation of the regulatory pathway that controls carotene biosynthesis was supported by the light-induced and photoadapted expression of all structural carotenogenic genes of B. trispora. Consequently, the beta-carotene content of dark grown mycelia of B. trispora increased upon illumination with white light.     

A RING-finger protein regulates carotenogenesis via proteolysis-independent ubiquitylation of a white collar-1-like activator

Protein ubiquitylation plays a major role in the regulation of cellular processes mainly through proteasome-dependent degradation, although it has become increasingly clear that it is also involved in other processes. In the fungus Mucor circinelloides, blue light regulates carotene biosynthesis, with this response controlled by crgA and mcwc-1c genes. CrgA shows characteristics of ubiquitin ligases and represses carotenogenesis in the dark, whereas mcwc-1c is a white collar 1-like gene required for its light induction. Another two white collar 1-like genes have been identified in M. circinelloides: mcwc-1a, which is involved in phototropism, and mcwc-1b, of unknown function. Analysis of double knockout mutants generated for crgA and every mcwc-1 gene demonstrated that crgA and mcwc-1c regulate carotenogenesis by independent pathways. It was also shown that the effect of crgA on carotenogenesis is mediated by mcwc-1b, which acts as a carotenogenesis activator. CrgA is involved in proteolysis-independent mono- and di-ubiquitylation of MCWC-1b, which results in its inactivation. Regulation of carotenogenesis in M. circinelloides by proteolysis-independent ubiquitylation suggests that this mechanism of control could be more widespread than previously thought.     

A RING-finger photocarotenogenic repressor involved in asexual sporulation in Mucor circinelloides.

Mucor circinelloides responds to blue light by activating the biosynthesis of carotenoids and bending its sporangiophores towards the light source. The CrgA protein product acts as a repressor of carotene biosynthesis, as its inactivation leads to the overaccumulation of carotenoids in both the dark and the light. We show here that asexual sporulation in Mucor is also stimulated by light and that the crgA gene is involved in sporulation, given that lack of crgA function affects both carotenogenesis and the normal production of spores. A small interference RNA (siRNA) gene silencing approach was used to block the biosynthesis of carotenoids and to demonstrate that abnormal sporulation in crgA mutants is not a consequence of a defective production of carotenes. These results reveal an active role for the predicted CrgA product, a RING-finger protein, in the control of cellular light-regulated processes in Mucor.     

Overexpression of the crgA gene abolishes light requirement for carotenoid biosynthesis in Mucor circinelloides.

This work describes the isolation and characterization of crgA, a Mucor circinelloides gene, which has a dominant-positive effect on light-regulated carotenogenesis. The crgA gene was originally identified in a transformation experiment as a 3'-truncated open reading frame which caused carotenoid overaccumulation in the dark. The complete cloning and sequencing of crgA revealed that its putative product presented several recognizable structural domains: a RING-finger zinc binding domain near the N-terminus, a putative nuclear localization signal, two stretches of acidic amino acids, glutamine-rich regions and a putative isoprenylation motif. The expression of exogenous copies of the complete crgA gene or two different 3'-truncated versions, produced a similar dominant-positive effect on the light-inducible carotenogenesis of M. circinelloides. The presence of these exogenous sequences also caused a missregulation of the endogenous crgA gene, resulting in its overexpression. Collectively, these observations suggest that crgA is involved in the regulation of carotenoid biosynthesis by light.     

The RING-finger domain of the fungal repressor crgA is essential for accurate light regulation of carotenogenesis

Mucor circinelloides responds to blue light by activating the biosynthesis of carotenoids. Gene crgA acts as a repressor of this light-regulated process, as its inactivation leads to overaccumulation of carotenoids in both the dark and the light. The predicted CrgA protein contains different recognizable structural domains, including a RING-finger zinc-binding motif, several glutamine-rich regions, a putative nuclear localization signal and an isoprenylation domain. To gain insight into the specific mode of action of the CrgA protein, we sought to define the CrgA domains critical for the light regulation of carotenogenesis. For this, mutant crgA alleles harbouring missense or deletion mutations in conserved residues of those domains were generated, and their functionality was assessed by testing their ability to complement a null crgA mutation. Point mutations of the amino-terminal RING-finger domain abrogated the ability of CrgA to repress carotenogenesis in the dark, as did the deletion of a poly glutamine-rich region at the carboxyl domain of CrgA. In contrast, mutations of the isoprenylation domain only slightly affected the CrgA function in carotenogenesis. The results identify two functional domains presumably involved in protein-protein interaction in the CrgA protein and suggest a role for the ubiquitin-proteasome pathway in the light regulation of carotenogenesis in fungi.     

Expression of the carotenoid biosynthesis genes in Xanthophyllomyces dendrorhous

In the yeast Xanthophyllomyces dendrorhous the genes idi, crtE, crtYB, crtl and ast are involved in the biosynthesis of astaxanthin from isopentenyl pyrophosphate. The carotenoid production and the kinetics of mRNA expression of structural genes controlling the carotenogenesis in a wild-type ATCC 24230 and in carotenoid overproducer deregulated atxS2 strains were studied. The biosynthesis of carotenoid was induced at the late exponential growth phase in both strains. However, the cellular carotenoid concentration was four times higher in atxS2 than in the wild-type strain in the exponential growth phase, suggesting that carotenogenesis was deregulated in atxS2 at the beginning of growth. In addition, the maximum expression of the carotenogenesis genes at the mRNA level was observed during the induction period of carotenoid biosynthesis in the wild-type strain. The mRNA level of the crtYB, crtl, ast genes and to a lesser extent the idi gene, decayed at the end of the exponential growth phase. The mRNA levels of the crtE gene remained high along the whole growth curve of the yeast. In the atxS2 strain the mRNA levels of crtE gene were about two times higher than the wild-type strain in the early phase of the growth cycle.     

Distinct white collar-1 genes control specific light responses in Mucor circinelloides

Light regulates many developmental and physiological processes in a large number of organisms. The best-known light response in the fungus Mucor circinelloides is the biosynthesis of beta-carotene. Here, we show that M. circinelloides sporangiophores also respond to light, exhibiting a positive phototropism. Analysis of both responses to different light wavelengths within the visible spectrum demonstrated that phototropism is induced by green and blue light, whereas carotenogenesis is only induced by blue light. The blue regulation of both responses suggests the existence of blue-light photoreceptors in M. circinelloides. Three white collar-1 genes (mcwc-1a, mcwc-1b and mcwc-1c) coding for proteins showing similarity with the WC-1 photoreceptor of Neurospora crassa have been identified. All three contain a LOV (light, oxygen or voltage) domain, similar to that present in fungal and plant blue-light receptors. When knockout mutants for each mcwc-1 gene were generated to characterize gene functions, only mcwc-1c mutants were defective in light induction of carotene biosynthesis, indicating that mcwc-1c is involved in the light transduction pathway that control carotenogenesis. We have also shown that positive phototropism is controlled by the mcwc-1a gene. It seems therefore that mcwc-1a and mcwc-1c genes control different light transduction pathways, although cross-talk between both pathways probably exists because mcwc-1a is involved in the light regulation of mcwc-1c expression.     

Photoinduction of carotenoid biosynthesis in Gibberella fujikuroi

Abstract Carotenoid biosynthesis is photoinducible in Gibberella fujikuroi , an organism used in the fermentive production of the gibberellins. The light exposed needed for an appreciable response is higher than those required for other fungi, such as Fusarium aquaeductuum and Neurospora crassa , under identical conditions. Time course of the accumulation of carotenoids is very similar to that for Fusarium aquaeductuum . Growth in one of the culture media used increases the carotenoid content in the dark but does not affect photoinduction. Three mutants with enhanced carotenoid synthesis in the dark show the same response to light as the wild-type. Our results suggest that photoinduction of carotenogenesis in Gibberella fujikori is independent of the carotenoid content already present in dark-grown cultures.     

Carotene-superproducing strains of Phycomyces.

Production of beta-carotene by wild-type Phycomyces blakesleeanus can be stimulated by light, chemicals, regulatory mutations, and sexual interaction between mycelia of opposite sex. Through genetic manipulations, we have isolated strains which have simultaneously and constitutively incorporated several of these stimulatory effects. In the dark and in a simple medium, some of the strains produce up to 25 mg of beta-carotene per g (dry weight), or about 500 times the wild-type production under the same conditions. High lycopene-producing strains have also been isolated by using carR mutants, which are blocked in the conversion of lycopene to beta-carotene. These strains should be useful in both industrial production of these pigments and basic research related to carotenogenesis.     

End-product regulation of carotenogenesis in Phycomyces

Wild-type Phycomyces blakesleeanus synthesizes the yellow pigment, beta-carotene. Colour mutants exhibit various alterations in the biosynthesis of beta-carotene or in its regulation. The presence of certain chemicals in the medium stimulates carotenogenesis in the wild type. We attribute different mechanisms of action to agents which stimulate or fail to stimulate different sets of mutants; this is the case of retinol and dimethyl phthalate. Dimethyl phthalate and veratrol are active on the same mutants, and therefore are likely to act in the same way. The main regulation of carotenogenesis, end-product inhibition, does not operate in the mutants of certain genes; these mutants are indifferent to retinol. By using a collection of retinoids we conclude that their action depends on their structural similarity to a part of the beta-carotene molecule. From these and other observations we propose that end-product inhibition of the pathway is mediated by a complex of beta-carotene and two gene products and that the retinoids compete with beta-carotene and prevent end-product inhibition.Deceased     

Regulation of Carotenogenesis and Secondary Metabolism by Nitrogen in Wild-Type Fusarium fujikuroi and Carotenoid-Overproducing Mutants

The fungus Fusarium fujikuroi (Gibberella fujikuroi MP-C) produces metabolites of biotechnological interest, such as gibberellins, bikaverins, and carotenoids. Gibberellin and bikaverin productions are induced upon nitrogen exhaustion, while carotenoid accumulation is stimulated by light. We evaluated the effect of nitrogen availability on carotenogenesis in comparison with bikaverin and gibberellin production in the wild type and in carotenoid-overproducing mutants (carS). Nitrogen starvation increased carotenoid accumulation in all strains tested. In carS strains, gibberellin and bikaverin biosynthesis patterns differed from those of the wild type and paralleled the expression of key genes for both pathways, coding for geranylgeranyl pyrophosphate (GGPP) and kaurene synthases for the former and a polyketide synthase for the latter. These results suggest regulatory connections between carotenoid biosynthesis and nitrogen-controlled biosynthetic pathways in this fungus. Expression of gene ggs1, which encodes a second GGPP synthase, was also derepressed in the carS mutants, suggesting the participation of Ggs1 in carotenoid biosynthesis. The carS mutations did not affect genes for earlier steps of the terpenoid pathway, such as fppS or hmgR. Light induced carotenoid biosynthesis in the wild type and carRA and carB levels in the wild-type and carS strains irrespective of nitrogen availability.     

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.