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.

Apocarotenoids produced from β-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.

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].

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

The oxidative cleavage of β-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 β-carotene. In cultures of Phycomyces blakesleeanus we detected thirty-two apocarotenoids by LC, UV absorbance, and MS.

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.

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

The Mucoral fungus Blakeslea trispora is used for the industrial production of β-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.

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.

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).

New apocarotenoids and β-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.

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 β-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.

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.

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.

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.

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.

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.

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.

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.