Functions of polyamines in growth and development of Phycomyces blakesleeanus wild-type and mutant strains.

Polyamines are ubiquitous polycationic molecules with multiple effects. Spermidine was present in all the life stages of Phycomyces blakesleeanus, fulfilled the physiological requirement for polyamines during germination, and became most abundant at the emergence of germinating tubes. Putrescine was not found in resting spores or in stationary cultures, but was synthesized during apical growth and greatly exceeded spermidine in fast-growing stages of the vegetative and sexual life cycles. Changes in the polyamines did not correlate with the various stages of sporulation. Ornithine decarboxylase was so strongly inhibited in vitro by its product, putrescine, that it would completely block the enzyme if not compartmentalized away. 1,4-Diamino-2-butanone inhibited mycelial growth throughout the vegetative cycle without killing the cells. The inhibition was counteracted very effectively by putrescine, which acts as a close analog of the inhibitor, and very little by spermidine. Four independent spe mutants were obtained by a procedure that selected for resistance to diaminobutanone among functionally-uninucleate spores that survived exposure to N-methyl-N'-nitro-N-nitrosoguanidine. The stability of the enzyme, in vivo and in vitro, and its inhibition by diaminobutanone in vitro were the same in the wild type and in the mutants. Two of these were hypomorph mutants, with lower affinity of their ornithine decarboxylase for its substrate, ornithine, and lower maximal velocity. The other two were hypermorph transport mutants; we propose that they are affected in a protein that binds putrescine and its analogs for transport across the plasmalemma and sequestration away from the active enzyme. The transport mutants concentrated the exogenous diaminobutanone and the endogenous putrescine in inactive compartments; the highest enzyme activity was reached when the plasmalemma of the mutants was permeabilized with diethylaminoethyl dextran.

Apocarotenoids produced from ß-carotene by dioxygenases from Mucor circinelloides.

Mucor circinelloides exhibits the complex sexual behaviour that is induced in other Mucoromycotina by a family of apocarotenoids called trisporoids. The genome of M. circinelloides contains four genes encoding putative carotenoid cleavage dioxygenases. The gene products of two of them were sufficient to convert ß-carotene into the precursors of three families of apocarotenoids, both in vitro and in the Escherichia coli heterologous in vivo system. The first of these products, CarS, cleaved the C40 ß-carotene into the C15 precursor of cyclofarnesoids and a C25 apocarotenal that was converted by the second enzyme, AcaA, into the C18 precursor of trisporoids and the C7 precursor of methylhexanoids. Apocarotenoids were not found in single or mixed cultures of the two strains of opposite sex, whose interaction readily produced zygospores, the sexual fusion cells.

Expansion of Signal Transduction Pathways in Fungi by Extensive Genome Duplication.

Plants and fungi use light and other signals to regulate development, growth, and metabolism. The fruiting bodies of the fungus Phycomyces blakesleeanus are single cells that react to environmental cues, including light, but the mechanisms are largely unknown [1]. The related fungus Mucor circinelloides is an opportunistic human pathogen that changes its mode of growth upon receipt of signals from the environment to facilitate pathogenesis [2]. Understanding how these organisms respond to environmental cues should provide insights into the mechanisms of sensory perception and signal transduction by a single eukaryotic cell, and their role in pathogenesis. We sequenced the genomes of P. blakesleeanus and M. circinelloides and show that they have been shaped by an extensive genome duplication or, most likely, a whole-genome duplication (WGD), which is rarely observed in fungi [3-6]. We show that the genome duplication has expanded gene families, including those involved in signal transduction, and that duplicated genes have specialized, as evidenced by differences in their regulation by light. The transcriptional response to light varies with the developmental stage and is still observed in a photoreceptor mutant of P. blakesleeanus. A phototropic mutant of P. blakesleeanus with a heterozygous mutation in the photoreceptor gene madA demonstrates that photosensor dosage is important for the magnitude of signal transduction. We conclude that the genome duplication provided the means to improve signal transduction for enhanced perception of environmental signals. Our results will help to understand the role of genome dynamics in the evolution of sensory perception in eukaryotes.

Cyclofarnesoids and methylhexanoids produced from ß-carotene in Phycomyces blakesleeanus.

The oxidative cleavage of ß-carotene in the Mucorales produces three fragments of 18, 15, and 7 carbons, respective heads of three families of apocarotenoids: the methylhexanoids, the trisporoids, and the cyclofarnesoids (named after their 1,6-cyclofarnesane skeleton). The apocarotenoids are easily recognized because they are absent in white mutants unable to produce ß-carotene. In cultures of Phycomyces blakesleeanus we detected thirty-two apocarotenoids by LC, UV absorbance, and MS. With additional IR and NMR we identified two methylhexanoids and the eight most abundant cyclofarnesoids. Four of them were previously-unknown natural compounds, including 4-dihydrocyclofarnesine S, the most abundant cyclofarnesoid in young cultures. We arranged the apocarotenoids of the Mucorales in a scheme that helps classifying and naming them and suggests possible metabolites and biosynthetic pathways. We propose specific biosynthetic pathways for cyclofarnesoids and methylhexanoids based on structural comparisons, the time course of appearance of individual compounds, and the bioconversion of ß-apo-12-carotenol, an early precursor, to three more oxygenated cyclofarnesoids by the white mutants. Some of the reactions occur spontaneously in the increasingly acidic culture media. Mating increased the contents of methylhexanoids and cyclofarnesoids by ca. threefold in young cultures and ca. twelvefold in old ones (five days); cyclofarnesine S, the most abundant cyclofarnesoid in old cultures, increased over one hundredfold. We found no differences between the sexes and no activity as sexual pheromones, but we suggest that methylhexanoids and cyclofarnesoids could mediate species-specific physiology and behavior.

Diversity, ecology, and evolution in Phycomyces.

The fungal genus Phycomyces (Mucoromycotina, Mucorales) has been revised by examining 96 strains, received from established collections or newly isolated from different environments. Morphology, sexuality, DNA sequences, and population structure clearly identify the genus and set it apart from other Mucorales. The size of the spores, the sexual interactions, the sequences of genes sexM and sexP that determine sexual identity, and the DNA for ribosomal RNAs validate the species Phycomyces blakesleeanus and Phycomyces nitens and the family Phycomycetaceae. Isolates from the same sample of biomass often differ in spore size, sex, DNA sequences, and restriction-length polymorphisms. The widest diversity was found in similar environments in three of the Canary Islands, implying a failure to disperse rapidly over the sea. All strains have apparently functional sexM and sexP genes and all but some strains of P. nitens complete the sexual cycle in the laboratory. The genetic diversity of P. blakesleeanus strains provides evidence for geographical clustering. Various sequence comparisons, including the newly isolated genes sexM and sexP of P. nitens and Blakeslea trispora, clarify phylogenetic relationships in the Mucorales and recommend the sex genes for the study of speciation.

Isolation of mutants and construction of intersexual heterokaryons of Blakeslea trispora.

The Mucoral fungus Blakeslea trispora is used for the industrial production of ß-carotene and lycopene. Two genetic techniques have been used to increase carotene accumulation: the isolation of mutants and the formation and segregation of heterokaryons. Because all life stages are multinucleated, recessive mutants are isolated after exposure to N-methyl-N'-nitro-N-nitrosoguanidine, a strong mutagen and inactivator of nuclei. Intersexual heterokaryons are obtained easily, because they are formed spontaneously during sexual interaction. Here are the pertaining methods, based on those previously developed for Phycomyces blakesleeanus, a related and better-known fungus.

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 ß-carotene. Phycomyces mutants in gene carS accumulate large amounts of ß-carotene but do not enter the sexual process. We show that carS encodes a ß-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 ß-ring-containing apocarotenoid that inhibits the activity of the carotenogenic enzyme complex in vegetative cells and provides a feedback regulation for the ß-carotene pathway. The carS gene product is a keystone in carotenogenesis and in sexual reproduction.

Apocarotenoids in the sexual interaction of Phycomyces blakesleeanus.

A simple genetic test allowed us to carry out the first systematic study of the apocarotenoids in the Mucorales. We have identified 13 apocarotenoids in the culture media of the fungus Phycomyces blakesleeanus (Mucoromycota, Mucorales). Three of these compounds were novel apocarotenoids: (2S,8R,E)-8,14-epoxycyclofarnesa-4,6,9-triene-2,11-diol (6), (2S,6E,8E)-cyclofarnesa-4,6,8-triene-2,10,11-triol (7), and its 6Z isomer (8). Four of the remaining compounds have been reported previously from this fungus and six from other Mucorales. All of them belong to three families, the 18-carbon trisporoids, the 15-carbon cyclofarnesoids, and the 7-carbon methylhexanoids, derived from the three fragments that result when ß-carotene is cleaved at its 11',12' and 12,13 double bonds. The apocarotenoids were more varied and more abundant in mated cultures of strains of opposite sex than in single cultures. The presence of acetate in the medium blocked the production of many apocarotenoids while having little effect on the concentrations of the remaining ones.

Cleavage oxygenases for the biosynthesis of trisporoids and other apocarotenoids in Phycomyces.

Mixed cultures of strains of opposite sex of the Mucorales produce trisporic acids and other compounds arising from cleavage of ß-carotene, some of which act as signals in the mating process. The genome of Phycomyces blakesleeanus contains five sequences akin to those of verified carotenoid cleavage oxygenases. All five are transcribed, three of them have the sequence traits that are considered essential for activity, and we have discovered the reactions catalysed by the products of two of them, genes carS and acaA. The transcripts of carS became more abundant in the course of mating, and its expression in ß-carotene-producing Escherichia coli cells led to the formation of ß-apo-12'-carotenal, a C25 cleavage product of ß-carotene. Joint expression of both genes in the same in vivo system resulted in the production of ß-apo-13-carotenone, a C18 fragment. In vitro, AcaA cleaved ß-apo-12'-carotenal into ß-apo-13-carotenone and was active on other apocarotenoid substrates. According to these and other results, the first reactions in the apocarotenoid pathway of Phycomyces are the cleavage of ß-carotene at its C11'-C12' double bond by CarS and the cleavage of the resulting C25-fragment at its C13-14 double bond by AcaA.

New apocarotenoids and ß-carotene cleavage in Blakeslea trispora.

Mixed cultures of strains of opposite sex ("mated" cultures) of Blakeslea trispora contain trisporic acids and other apocarotenoids, some of which mediate the sexual responses of this fungus and other Mucorales. In mated cultures of the wild-type strains F986 and F921 we identified eleven apocarotenoids: two C(18) trisporoids, three C(15) compounds with a monocyclofarnesane skeleton, a C(13) compound, and five C(7) compounds with a 2-methylhexane skeleton. Six of them are new natural products and two others are new for Blakeslea. Their structures were established by NMR and mass spectra and those of the C(7) and C(13) compounds were confirmed by chemical synthesis. The finding of these compounds and the presence of approximately equimolecular amounts of the C(18), C(15), and C(7) families led to the conclusion that ß-carotene is initially split in three fragments by cleavage of its 13,14 and 11',12' double bonds.

Splitting of beta-carotene in the sexual interaction of Phycomyces.

Two new 7-carbon compounds, 1 and 2, have been found in the culture medium of Phycomyces blakesleeanus. A genetic test showed that they derive from beta-carotene. These new molecules represent the missing link that proves that beta-carotene is split into fragments of 18, 15 and 7 carbon fragments, each head of a separate family of apocarotenoids.

Genes involved in carotene synthesis and mating in Blakeslea trispora.

Mating of Blakeslea trispora and other molds of the order Mucorales requires the interaction of mycelia of opposite sex, (+) and (-), leading to the development of specialized structures and to an enhanced accumulation of beta-carotene. Industry obtains beta-carotene by co-cultivating appropriate strains of Blakeslea ("mated cultures"). Gene transcription in single and mated cultures was assayed by cDNA-AFLP, a technique to observe the differential expression of subsets of mRNA fragments. Overexpression in mated cultures is about ten times more frequent than underexpression. We obtained and sequenced fragments of 97 candidate genes that appeared to be overexpressed during mating and confirmed four of them by reverse transcription and real-time PCR. Comparisons with gene sequences from other organisms suggest functions in carotene biosynthesis (4 genes), energy metabolism (8), cell wall synthesis (1), transfer of acetyl groups (1), and regulatory processes (10). Sodium acetate inhibited sexual overexpression in about two-thirds of the candidate genes and acted as a signal with broad effects on the metabolism and the morphology of mated cultures. Our work offers new materials for the study of carotene biosynthesis and its regulation and for the improvement of carotene production with Mucorales.

Gene expression in the regulation of carotene biosynthesis in Phycomyces.

Carotene synthesis in the Mucoral fungus, Phycomyces blakesleeanus, is regulated by a complex genetic mechanism and activated by four groups of environmental factors with independent mechanisms of action. Blue light and sexual stimulation increased in parallel the content of carotene and the content of mRNAs from the genes, carRA and carB, dedicated to the synthesis of beta-carotene from geranylgeranyl pyrophosphate. The effects of these agents were approximately additive. Retinol and dimethyl phthalate, which represent the remaining groups of activators, greatly increased the carotene content, but did not modify the levels of carRA and carB transcripts. Mutants in genes carRA, carB, carC, carD, carF, carI, and carS differed in their carotene content, from nil to much larger than that of the wild type, but had the same carRA and carB transcript levels as the wild type. The only exception was a carRA early-stop mutant, which had very small amounts of the carRA transcript. The genetic and environmental factors that modify carotene biosynthesis had little or no effect on the mRNA levels of genes, hmgS and hmgR, responsible for the enzymes that initiate the biosynthesis of all terpenoids. A general model for the regulation of carotenogenesis in Phycomyces was derived from the results.

Ubiquinone and carotene production in the Mucorales Blakeslea and Phycomyces.

The filamentous fungi Phycomyces blakesleeanus and Blakeslea trispora (Zygomycota, Mucorales) are actual or potential industrial sources of beta-carotene and lycopene. These chemicals and the large terpenoid moiety of ubiquinone derive from geranylgeranyl pyrophosphate. We measured the ubiquinone and carotene contents of wild-type and genetically modified strains under various conditions. Light slightly increased the ubiquinone content of Blakeslea and had no effect on that of Phycomyces. Oxidative stress modified ubiquinone production in Phycomyces and carotene production in both fungi. Sexual interaction and mutations in both organisms made the carotene content vary from traces to 23 mg/g dry mass, while the ubiquinone content remained unchanged at 0.3 mg/g dry mass. We concluded that the biosyntheses of ubiquinone and carotene are not coregulated. The specific regulation for carotene biosynthesis does not affect even indirectly the production of ubiquinone, as would be expected if terpenoids were synthesized through a branched pathway that could divert precursor flows from one branch to another.

Relationships among the biosyntheses of ubiquinone, carotene, sterols, and triacylglycerols in Zygomycetes.

The Zygomycetes Phycomyces blakesleeanus and Blakeslea trispora are actual or potential sources of beta-carotene, ergosterol, ubiquinone, edible oil, and other compounds. By feeding [14C]acetyl-CoA, L-[14C]leucine, or R-[14C]mevalonate in the presence of excess unlabeled glucose, we found that ubiquinone (the terpenoid moiety), beta-carotene, and triacylglycerols were made from separate pools of all their common intermediates; the pools for ubiquinone and ergosterol were indistinguishable. Fatty acids were not labeled from mevalonate, showing the absence in these fungi of a shunt pathway that would recycle carbon from mevalonate and its products back to central metabolism. The overproduction of carotene in a Phycomyces mutant and in sexually mated cultures of Blakeslea modified the relative use of labeled and unlabeled carbon sources in the production of carotene, but not of the other compounds. We concluded that carotene, ubiquinone, and triacylglycerols are synthesized in separate subcellular compartments, while sterols and ubiquinone are synthesized in the same compartments or in compartments that exchange precursors. Carotene biosynthesis was regulated specifically and not by flow diversion in a branched pathway.

Modification of sexual development and carotene production by acetate and other small carboxylic acids in Blakeslea trispora and Phycomyces blakesleeanus.

In Phycomyces blakesleeanus and Blakeslea trispora (order Mucorales, class Zygomycetes), sexual interaction on solid substrates leads to zygospore development and to increased carotene production (sexual carotenogenesis). Addition of small quantities of acetate, propionate, lactate, or leucine to mated cultures on minimal medium stimulated zygospore production and inhibited sexual carotenogenesis in both Phycomyces and Blakeslea. In Blakeslea, the threshold acetate concentration was <1 mmol/liter for both effects, and the concentrations that had one-half of the maximal effect were <2 mmol/liter for carotenogenesis and >7 mmol/liter for zygosporogenesis. The effects on Phycomyces were similar, but the concentrations of acetate had to be multiplied by ca. 3 to obtain the same results. Inhibition of sexual carotenogenesis by acetate occurred normally in Phycomyces mutants that cannot use acetate as a carbon source and in mutants whose dormant spores cannot be activated by acetate. Small carboxylic acids may be signals that, independent of their ability to trigger spore germination in Phycomyces, modify metabolism and development during the sexual cycle of Phycomyces and Blakeslea, uncoupling two processes that were thought to be linked and mediated by a common mechanism.

Genetic damage following introduction of DNA in Phycomyces.

Introduction of plasmids in Phycomyces blakesleeanus caused extensive changes in the exogenous DNA and in the resident genome. Plasmids with a bacterial gene for geneticin resistance under a Phycomyces promoter were either injected into immature sporangia or incubated with spheroplasts. An improved method produced about one viable spheroplast per cell. Colonies resistant to geneticin were rare and only about 0.1% of their spores grew in the presence of geneticin. The transformation frequency was very low, < or =1 transformed colony per million spheroplasts or per microg DNA. Few nuclei in the transformants contained exogenous DNA, as shown by a selective procedure that sampled single nuclei from heterokaryons. The exogenous DNA was not integrated into the genome and no stable transformants were obtained. The plasmids were replicated in the recipient cells, but their DNA sequences were modified by deletions and rearrangements and the transformed phenotype was eventually lost. The spores developed in injected sporangia were often inviable; a genetic test showed that spore death was caused by impaired nuclear proliferation and induction of lethal mutations. About one-fourth of the viable spores from injected sporangia formed abnormal colonies with obvious changes in shape, texture, or color. The abnormalities that could be investigated were due to dominant mutations. The results indicate that incoming DNA is not only attacked, but signals a situation of stress that leads to increased mutation and nuclear and cellular death.

Mutants and intersexual heterokaryons of Blakeslea trispora for production of beta-carotene and lycopene.

The industrial production of beta-carotene with the zygomycete Blakeslea trispora involves the joint cultivation of mycelia of opposite sex in the presence of beta-ionone and other chemical activators. We have obtained improved strains by mutation and heterokaryosis. We chose wild strains on the basis of their growth and carotene content in single and mated cultures. Following exposure of their spores to N-methyl-N'-nitro-N-nitrosoguanidine, we obtained high-carotene mutants, which were more productive than their parents but similar to them in having beta-carotene as the main product. Further increases in carotene content were obtained after a new round of mutagenesis in one of the mutants. The production was shifted to lycopene in cultures incubated in the presence of nicotine and in lycopene-rich mutants derived from the wild strains. The highest production levels were achieved in intersexual heterokaryons, which contained mutant nuclei of opposite sex. These contained up to 39 mg of beta-carotene or 15 mg of lycopene per g (dry mass) under standard laboratory conditions in which the original wild strains contained about 0.3 mg of beta-carotene per g (dry mass). Beta-ionone did not increase the carotene content of these strains. Not all wild strains lent themselves to these improvements, either because they produced few mutants or because they did not increase their carotene production in mated cultures.