Functional analysis of the Phycomyces carRA gene encoding the enzymes phytoene synthase and lycopene cyclase

Phycomyces carRA gene encodes a protein with two domains. Domain R is characterized by red carR mutants that accumulate lycopene. Domain A is characterized by white carA mutants that do not accumulate significant amounts of carotenoids. The carRA-encoded protein was identified as the lycopene cyclase and phytoene synthase enzyme by sequence homology with other proteins. However, no direct data showing the function of this protein have been reported so far. Different Mucor circinelloides mutants altered at the phytoene synthase, the lycopene cyclase or both activities were transformed with the Phycomyces carRA gene. Fully transcribed carRA mRNA molecules were detected by Northern assays in the transformants and the correct processing of the carRA messenger was verified by RT-PCR. These results showed that Phycomyces carRA gene was correctly expressed in Mucor. Carotenoids analysis in these transformants showed the presence of ß-carotene, absent in the untransformed strains, providing functional evidence that the Phycomyces carRA gene complements the M. circinelloides mutations. Co-transformation of the carRA cDNA in E. coli with different combinations of the carotenoid structural genes from Erwinia uredovora was also performed. Newly formed carotenoids were accumulated showing that the Phycomyces CarRA protein does contain lycopene cyclase and phytoene synthase activities. The heterologous expression of the carRA gene and the functional complementation of the mentioned activities are not very efficient in E. coli. However, the simultaneous presence of both carRA and carB gene products from Phycomyces increases the efficiency of these enzymes, presumably due to an interaction mechanism.     

Production of fatty acid methyl esters and other bioactive compounds in elicited cultures of the fungus <Emphasis Type="Italic">Mucor circinelloides</Emphasis>

In this work, the production of carotenoids and volatile compounds in Mucor circinelloides cell cultures treated with methyl jasmonate (MJ) and/or cyclodextrins (CD) was evaluated. CD increased the lutein concentration in the extracellular medium, reaching the highest levels in the combined treatment with MJ, whereas the levels of β-carotene were low. Therefore, the addition of CD to M. circinelloides cultures provokes a release of these compounds into the culture medium. Mucor circinelloides cultures also produced lichesterol, neoergosterol and ergone, suggesting that, under these stress conditions, this fungus diverts the carbon flow to sterol biosynthesis, which, in turn, is required for its survival. More interestingly, CD induced the secretion of sterols in a similar way to carotenoids. Mucor circinelloides cultures treated with MJ and/or CD also produced fatty acid methyl esters (FAMEs) and, in the presence of CD, they were released to culture medium, contributing to the formation of biodiesel. In this way, M. circinelloides cultures produced compounds of biotechnological interest and, therefore, these treated cultures can provide an alternative system, which is, at the same time, more sustainable, economical and ecological for their production.     

Analysis of proteomic changes in colored mutants of Xanthophyllomyces dendrorhous (Phaffia rhodozyma)

The yeast Xanthophyllomyces dendrorhous synthesizes astaxanthin as its most prevalent xanthophyll derivative. Comparisons between the protein profiles of mutant lines of this yeast can provide insight into the carotenogenic pathway. Differently colored mutants (red, orange, pink, yellow, and white) were obtained from this yeast species, and their protein profiles were determined using two-dimensional polyacrylamide gel electrophoresis (2DE). Individual proteins differentially expressed were identified using mass spectrometry. The red mutants hyperproduced total carotenoids (mainly astaxanthin), while in white and orange mutants, mutagenesis affected the phytoene dehydrogenase activity as indicated by the accumulation of phytoene. Inactivation of astaxanthin synthase after the mutagenic treatment was evident in β-carotene accumulating mutants. Differences in the proteomic profiles of wild-type X. dendrorhous and its colored mutants were demonstrated using 2DE. Of the total number of spots detected in each gel (297–417), 128 proteins were present in all strains. The red mutant showed the greatest number of matches with respect to the wild type (305 spots), while the white and yellow mutants, which had reduced concentrations of total carotenoids, presented the highest correlation coefficient (0.6) between each other. A number of differentially expressed proteins were sequenced, indicating that tricarboxylic acid cycle and stress response proteins are closely related to the carotenogenic process.     

A gene for carotene cleavage required for pheromone biosynthesis and carotene regulation in the fungus Phycomyces blakesleeanus

Mating and sexual development in fungi are controlled by molecular mechanisms that are specific for each fungal group. Mating in Phycomyces blakesleeanus and other Mucorales requires pheromones derived from β-carotene. Phycomyces mutants in gene carS accumulate large amounts of β-carotene but do not enter the sexual process. We show that carS encodes a β-carotene-cleaving oxygenase that catalyzes the first step in the biosynthesis of a variety of apocarotenoids, including those that act as pheromones. Therefore carS mutants cannot stimulate their sexual partners, although they respond to them. CarS catalyzes the biosynthesis of a β-ring-containing apocarotenoid that inhibits the activity of the carotenogenic enzyme complex in vegetative cells and provides a feedback regulation for the β-carotene pathway. The carS gene product is a keystone in carotenogenesis and in sexual reproduction.     

Activation of the biosynthesis of carotenoids by Blakeslea trispora

The research carried out by several scientists has made possible the industrial preparation of β-carotene by fermentation. A fungus, Blakeslea trispora, abundantly synthesizes carotenoids when its two opposite forms are cultivated together in a special fatty medium. When ionones or other natural substances are introduced into the culture, a very obvious increase in the biosynthesis of carotenoids, more specifically of β-carotene, is obtained. Our own work has shown that; (1) several synthetic products chemically related to β-ionone, such as 2,6,6-trimethyl-l-acetyleyelohexene, can advantageously replace either partially or totally the ionones as inductors of the biosysnthesis of β-carotene; (2) various nitrogen-containing substances when added to the culture medium can considerably enhance the biosysnthesis of carotenoids while sometimes very specically orienting it. Their action comes on top of that of the ionones or their substitutes; actually this action is unexplained. Thus certain amides, imides, lactams, hydrazides, or substituted pyradines, and in particular succinimide and isonicotinoylhydrazine, have produced a two or threefold increase in the quantity of β-carotene present in the culture media of Blackeslea trispora. Conversely some heterocyclic substances such as pyridine itself or imidazole totally inhibit the biosysnthesis of β-carotene but induce the production of very important quantities of lycopene.     

Simultaneous enrichment of cereals with polyunsaturated fatty acids and pigments by fungal solid state fermentations

Four Mucor strains were tested for their ability to grow on four cereal substrates and enriched them with gamma-linolenic acid (GLA) and β-carotene. M. circinelloides CCF-2617 as the best producer accumulated of both GLA and β-carotene in high amounts during utilization of rye bran/spent malt grains (3:1). The first growth phase was characterized by rapid GLA biosynthesis, while distinct β-carotene formation was found in the stationary fungal growth. Therefore various cultivation conditions were tested in order to optimize the yield of either GLA or β-carotene. The fungus grown on cereal substrate supplemented with glucose produced maximal 8.5 mg β-carotene and 12.1 g GLA in 1 kg fermented substrate, respectively. On the other hand, the highest amount of GLA in the fermented substrate (24.2 g/kg) was achieved when 30% of sunflower oil was employed to the substrate. Interestingly, β-carotene biosynthesis was completely inhibited when either whey or linseed oil were added to the substrate.     

Carotene-superproducing mutants ofPhycomyces blakesleeanus

The wild-type mycelia of the fungusPhycomyces blakesleeanus are yellow because they contain small amounts of β-carotene. Some mutations lead to large increases in β-carotene content. These “deep yellow” mutants carry recessive mutations in either of two genes,carD andcarS, not linked to each other or to other genes related to carotenogenesis. ThecarS mutants contain up to 100 times more β-carotene than the wild type; thecarD mutants, up to 20 times.carS mutants are unable to form zygospores and their carotenogenesis is not activated by retinol; on the other hand,carD mutants complete the sexual cycle and respond to retinol.carS mutations are epistatic overcarD mutations. The product of genecarS mediates the end-product regulation of the pathway; it is suggested that thecarD gene product increases the amount or the activity of thecarS gene product.     

Carotene biosynthesis by cell extracts of mutants of Phycomyces blakesleeanus

Cell extracts capable of converting MVA-[2-¹⁴C] into isoprenoids were obtained from the yellow C115-mad-107(−) and red C9-carR21(−) mutants of Phycomyces blakesleeanus. Neither air nor light was essential for carotene biosynthesis. The specific activities of the terpenoid-synthesizing enzymes varied with the age of the cultures although the formation of lycopene (ψ,ψ-carotene) in the C9 and of β-carotene (β,β-carotene) in the C115 'mutants. respectively, followed the increase in the dry weight yield of the cultures. The significance of these results to the biosynthesis of carotenes and to the classification of these compounds as secondary metabolites is discussed.     

Carotenoid oxidative degradation products inhibit Na+-K+-ATPase

This study investigates the biological significance of carotenoid oxidation products using inhibition of Na⁺-K⁺-ATPase activity as an index. β-Carotene was completely oxidized by hypochlorous acid and the oxidation products were analyzed by capillary gasliquid chromatography and high performance liquid chromatography. The Na⁺-K⁺-ATPase activity was assayed in the presence of these oxidized carotenoids and was rapidly and potently inhibited. This was demonstrated for a mixture of β-carotene oxidative breakdown products, β-Apo-10′-carotenal and retinal. Most of the β-carotene oxidation products were identified as aldehydic. The concentration of the oxidized carotenoid mixture that inhibited Na⁺-K⁺-ATPase activity by 50% (IC₅₀) was equivalent to 10μM non-degraded β-carotene, whereas the IC₅₀ for 4-hydroxy-2-nonenal, a major lipid peroxidation product, was 120 μM. Carotenoid oxidation products are more potent inhibitors of Na⁺-K⁺-ATPase than 4-hydroxy-2-nonenal. Enzyme activity was only partially restored with hydroxylamine and/or β-mercaptoethanol. Thus, in vitro binding of carotenoid oxidation products results in strong enzyme inhibition. These data indicate the potential toxicity of oxidative carotenoid metabolites and their activity on key enzyme regulators and signal modulators.     

Critical applications of Mucor circinelloides within a biorefinery context

The establishment of an efficient and feasible biorefinery model depends on, among other factors, particularly the selection of the most appropriate microorganism. Mucor circinelloides is a dimorphic fungus species able to produce a wide variety of hydrolytic enzymes, lipids prone to biodiesel production, carotenoids, ethanol, and biomass with significant nutritional value. M. circinelloides also has been selected as a model species for genetic modification by being the first filamentous oleaginous species to have its genome fully characterized, as well as being a species characterized as a potential bioremediation agent. Considering the potential of replacing several nonrenewable feedstocks is widely dependent on fossil fuels, the exploitation of microbial processes and products is a desirable solution for promoting a green and sustainable future. Here, we introduce and thoroughly describe the recent and critical applications of this remarkable fungus within the context of developing a fungal-based biorefinery.     

Cloning and characterization of genes involved in nostoxanthin biosynthesis of Sphingomonas elodea ATCC 31461

Most Sphingomonas species synthesize the yellow carotenoid nostoxanthin. However, the carotenoid biosynthetic pathway of these species remains unclear. In this study, we cloned and characterized a carotenoid biosynthesis gene cluster containing four carotenogenic genes (crtG, crtY, crtI and crtB) and a β-carotene hydroxylase gene (crtZ) located outside the cluster, from the gellan-gum producing bacterium Sphingomonas elodea ATCC 31461. Each of these genes was inactivated, and the biochemical function of each gene was confirmed based on chromatographic and spectroscopic analysis of the intermediates accumulated in the knockout mutants. Moreover, the crtG gene encoding the 2,2'-β-hydroxylase and the crtZ gene encoding the β-carotene hydroxylase, both responsible for hydroxylation of β-carotene, were confirmed by complementation studies using Escherichia coli producing different carotenoids. Expression of crtG in zeaxanthin and β-carotene accumulating E. coli cells resulted in the formation of nostoxanthin and 2,2'-dihydroxy-β-carotene, respectively. Based on these results, a biochemical pathway for synthesis of nostoxanthin in S. elodea ATCC 31461 is proposed.     

The effect of CPTA analogs and other nitrogenous compounds on the biosynthesis of carotenoids in Phycomyces blakesleeanus mutants

The inhibitory effect of a series of analogs of CPTA, 2-(4-chlorophenylthio)-triethylamine-HCl, and ammonia derivatives on carotenoid biosynthesis in Phycomyces blakesleeanus mutants was studied. The types of inhibition exhibited allowed no firm conclusions about the biosynthetic route to β-carotene from either β-zeacarotene or lycopene. However, the evidence suggests at present that both pathways are operative. It was found that a slight change in structure of inhibitor resulted in a different type of action. Conclusions based on a single inhibitor could be cited as “evidence” for a certain pathway.     

Interallelic complementation at the pyrF locus and the homodimeric nature of orotate phosphoribosyltransferase (OPRTase) in Mucor circinelloides

Using 5-fluoroorotic acid (5-FOA) as a positive selection system we isolated mutants of Mucor circinelloides altered in the pyrimidine biosynthetic pathway. These mutants were found to be deficient either in orotidine-5′-monophosphate decarboxylase (OMPdecase), or in orotate phosphoribosyltransferase (OPRTase) activity. Complementation tests among mutants lacking OPRTase activity classified them into three groups, thus suggesting the possibility of interallelic complementation. To investigate this hypothesis a cDNA clone corresponding to the OPRTase-encoding gene of M. circinelloides was isolated by direct complementation of E. coli. The genomic copy transformed to prototrophy one member of each of the three classes of OPRTase-deficient mutants. We therefore concluded that they were all altered at the same locus, the pyrF locus. The corresponding alleles were cloned and sequenced. Comparisons of the amino acid sequence of M. circinelloides OPRTase with those of E. coli and S. typhimurium revealed a high degree of similarity in secondary and tertiary structure. As the two bacterial enzymes exist as dimers, a homodimeric quaternary structure of the M. circinelloides mature protein can be assumed. This would also explain the interallelic complementation between some pyrF mutants. The mutations found could affect either the active site or the structure of the dimer interface of the OPRTase. Received: 22 May 1998 / Accepted: 13 August 1998     

Mutants of carotene production in Blakeslea trispora

The filamentous fungus Blakeslea trispora, an industrial carotene source, contains -carotene and precursors of its synthesis — phytoene, phytofluene, lycopene, and -carotene. Strain improvement through mutagenesis is difficult because all life stages are multinucleate. Mutants have been obtained following exposure of wild-type spores to N-methyl-N-nitro-N-nitrosoguanidine. Changes in the colour of the mycelia reflect variations in the accumulation of various precursors and the final product. Quantitative analysis of the mutants leads to the conclusion that the biosynthetic pathway is similar to that of the related fungus Phycomyces blakesleeanus, but the regulation is completely different. In particular, interruption of the pathway does not lead to overacummulation of precursors.     

Effects of light intensity and quality on the growth rate and photosynthetic pigment content of Spirulina platensis

The quantitative and qualitative effects of light on carotenoid production by Spirulina were studied. Maximum total carotenoid production was measured in cells grown under white light at an irradiance of 432 μmol photon m^?2 s^?1, the onset of light saturation for this organism as determined by growth rates. A true maximum may exist at irradiances above 1500 μmol photon m^?2 s^?1 under white light. Individual carotenoids responded differently to light conditions. Under white light, β-carotene and echinenone were most abundant at the lowest and highest irradiance levels tested. Myxoxanthophyll and lutein/zeaxanthin did not change over the same irradiance range. Under red and blue light, we found decreased values of myxoxanthophyll, while β-carotene increased and lutein/zeaxanthin and echinenone showed little change. In general, maximum carotenoid production requires optimization of the culture conditions that favor growth.     

Macromolecular Physiology of Plastids

The composition and amount of carotenoid pigments were determined in etiolated seedling leaves of 6 barley (Hordeum vulgare L.) mutants, comprising 1 xantha and 5 tigrina mutants. All mutants had on a mole basis approximately the same content of carotenoids as the wild type. The mutants xan-u²¹, tig-n³², and tig-³³ contained significantly higher amounts of carotenes than the wild type, ranging from 32 to 68% of the total carotenoid content as compared to the 4–8% found in the wild type. In the mutants tig-b²³ and tig-o³⁴, only a slight increase in the amount of carotenes was notable. The carotene content and composition in tig-d¹² was indistinguishable from that of the wild type. The carotenes extracted from xan-u²¹, tig-b²³, tig-n³², tig-³³, and tig-o³⁴ were characterized by adsorption chromatography and spectrophotometry. Mutant xan-u²¹ is in the dark blocked in β-carotene synthesis, and accumulates the aliphatic polyenes: phytofluene, proneurosporene, poly-cis-lycopenes, neo-lycopene and lycopene. The other four mutants synthesize β-carotene, but accumulate in addition various higher saturated carotenes, the main components being ζ-carotene in tig-b²³, a lycopenic pigment in tig-n³² and tig-³³, and lycopene in tig-o³⁴. Accumulation of higher saturated carotenes appears correlated with specific aberrations of the membrane structure in plastids. The regulation of carotene and protochlorophyllide syntheses in etioplasts are closely linked as shown by the single gene mutants which affect both pathways. However, several mutants have been identified which cause defects in protochlorophyllide synthesis only.     

Carotenogenesis in Phycomyces

Time course studies of carotenoid production and of mycelial growth in liquid cultures of Phycomyces blakesleeanus wild type [NRRL 1555 (?)], red mutants C9, C10 and C13 and the heterokaryon C2 * C9 are reported. The ratios of the concentrations of lycopene, γ-carotene and β-carotene in the red mutant C13 and in the heterokaryon C2 * C9 during the growth periods were measured. In these strains the concentration of lycopene is close to its final value after 2 days of growth, at a time at which β-carotene is just beginning to be produced. It is suggested that the β-carotene produced late is possibly synthesized via β-zeacarotene.     

Conversion of β-carotene into astaxanthin: Two separate enzymes or a bifunctional hydroxylase-ketolase protein?

Astaxanthin is a xanthophyll of great interest in animal nutrition and human health. The market prospect in the nutraceutics industries for this health-protective molecule is very promising. Astaxanthin is synthesized by several bacteria, algae and plants from β-carotene by the sequential action of two enzymes: a β-carotene, 3,3'-hydroxylase that introduces an hydroxyl group at the 3 (and 3') positions of each of the two β-ionone rings of β-carotene, and a β-carotene ketolase that introduces keto groups at carbons 4 and 4' of the β-ionone rings. Astaxanthin is also produced by the yeast-like basidiomycete Xanthophyllomyces dendrorhous. A gene crtS involved in the conversion of β-carotene to astaxanthin has been cloned simultaneously by two research groups. Complementation studies of X. dendrorhous mutants and expression analysis in Mucor circinelloides reveals that the CrtS enzyme is a β-carotene hydroxylase of the P-450 monooxygenase family that converts β-carotene to the hydroxylated derivatives β-cryptoxanthin and zeaxanthin, but it does not form astaxanthin or the ketolated intermediates in this fungus. A bifunctional β-carotene hydroxylase-ketolase activity has been proposed for the CrtS protein. The evidence for and against this hypothesis is analyzed in detail in this review.     

THE WAVELENGTH DEPENDENCE OF MASSIVE CAROTENE SYNTHESIS IN DUNALIELLA BARDAWIL (CHLOROPHYCEAE)1

Dunaliella bardawil Ben-Amotz & Avron accumulates high concentrations of β-carotene when grown under high light intensity. The β-carotene is composed mainly of 9-cis and all-trans β-carotene. Accumulation of β-carotene and an increase in the ratio of the 9-cis to the all-trans isomer are strongly dependent on the light intensity under which the algae are cultivated but are independent of light quality within the photosynthetically active radiation range. Cells grown under continuous red (>645 nm) or white light of 500 W·m^?2 reach a value of about 32 pg β-carotene·cell^?1 and a ratio of 9-cis to all-trans β-carotene of around 2, whereas cells grown under low red or white light intensity of 25 W·m^?2 contain about 3 pg·cell^?1 and a ratio of isomers of around 0.3.