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.     

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.     

A review of factors affecting biosynthesis of carotenoids by the order Mucorales

This is a review of factors affecting carotenogenesis by the order Mucorales which includes Phycomyces blakesleeanus, Choanephora cucurbitarum and Blakeslea trispora. The Mucorales have opposite sex types and when mated, -carotene production is increased 15 to 20 times. Trisporic acids are the substances produced upon mating which stimulate carotenogenesis. Structural analogs have been shown to mimic the actions of the trisporic acids. The common denominator of the stimulators is the ionone ring and the hydrocarbon side chain. Secondary metabolism is discussed as well as the use of food byproducts to stimulate, specifically, the production of -carotene by B. trispora.     

The correlation between the synthesis of beta-carotene and zygote formation by Blakeslea trispora heterothallic strains

It was demonstrated for the first time that the level of carotenogenesis by the heterothallic Blakeslea trispora strains intensively forming zygospores decreased under conditions of a surface cocultivation during their sexual interaction as compared with the strains grown separately. On the contrary, carotenogenesis was stimulated during a sexual interaction of the strains incapable of forming zygotes. In a submerged culture, the zygote-forming pairs of strains synthesized a considerably larger amount of trisporic acids but a smaller amount of carotenoids than the strains not forming zygospores. The discovered inverse dependence between zygote formation and carotenogenesis allowed us to suggest the inability to form zygotes as a criterion for selecting carotenogenic strain pairs.     

The correlation between the synthesis of β-carotene and zygote formation by <Emphasis Type="Italic">Blakeslea trispora</Emphasis> heterothallic strains

It was demonstrated for the first time that the level of carotenogenesis by the heterothallic Blakeslea trispora strains intensively forming zygospores decreased under conditions of a surface cocultivation during their sexual interaction as compared with the strains grown separately. On the contrary, carotenogenesis was stimulated during a sexual interaction of the strains incapable of forming zygotes. In a submerged culture, the zygote-forming pairs of strains synthesized a considerably larger amount of trisporic acids but a smaller amount of carotenoids than the strains not forming zygospores. The discovered inverse dependence between zygote formation and carotenogenesis allowed us to suggest the inability to form zygotes as a criterion for selecting carotenogenic strain pairs.     

Regulation of carotene synthesis in Phycomyces

Summary Threeindependent mutations of Phycomyces blakesleeanus resulting in overaccumulation of -carotene are recessive and belong to the same complementation group. The corresponding gene has been named carS. Evidence is presented that gene carS is not the same as gene carA, previously defined by mutations blocking carotene production. Vitamin A increases carotenogenesis in wild types and in carS mutants to about the same extent. Intersexual heterokaryosis increases carotenogenesis most prominently in carS genetic backgrounds (up to 300 times the production of the wild type in the same conditions). Vitamin A, intersexual heterokaryosis and carS mutations are thought to stimulate carotenogenesis through different mechanisms. It is suggested that the carS gene product participates in end-product regulation of the pathway.     

Isolation of new white collar mutants of Neurospora crassa and studies on their behavior in the blue light-induced formation of protoperithecia

White collar (wc) mutants of Neurospora crassa are thought to be regulatory mutants blocked in the photoinduction of carotenogenesis. Eight new wc mutants have been isolated after UV mutagenesis; their morphology and linear growth rate are not altered, although blue light-induced carotenogenesis is completely blocked. All of the wc mutations fall into two complementation groups corresponding to the already-known wc-1 and wc-2 loci. It is shown that the wc mutations impair another blue light effect, the photoinduction of protoperithecia formation, as well as the low constitutive production of protoperithecia in the dark. These effects are not due to the lack of carotenoids since the albino mutants show a normal sexual development. The pleiotropic effects of the mutations in the wc genes indicate that they play a key role in the mechanisms of regulation of the blue light-induced responses of N. crassa.     

Genetic determination of sporangiophore development in Phycomyces

The mycelium of the fungus Phycomyces. essentially a giant multinucleate cell, produces two kinds of asexual reproductive structures, called macrophores and microphores, and a succession of structures for sexual reproduction. Following the treatment of spores with N-methyl-N′ -nitro-N-nitrosoguanidine, conditional imb mutants have been isolated that form no macrophores at 26°C, but do at 14°C. At the restrictive temperature, few imb mutants (2 of 13) develop microphores, and none is able to complete the sexual cycle. This suggests that genes responsible for macrophorogenesis are involved in microphorogenesis and in sexual development as well. Light reduces macrophorogenesis and totally abolishes microphorogenesis in the wild type under the conditions of our experiments. These photomorphogenetic effects require the normal function of genes madA and madB, which are responsible for phototropism. Light inhibits microphorogenesis in the two imb mutants that form microphores at the restrictive temperature. Genetic alterations of carotenogenesis lead to an excess of microphores and a scarcity of macrophores in the dark, but they have little influence on vegetative reproduction in the light.     

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.     

The effect of veratrole on carotenoid biosynthesis by Phycomyces blakesleeanus

A number of chemical compounds are known to affect the biosynthetic pathways of β-carotene. Both site-specific inhibitors as well as general stimulators of carotenogenesis have been described. It has been reported that veratrole enhances β-carotene synthesis when applied to agar cultures of Phycomyces blakesleeanus but we found no significant stimulation of β-carotene production in submerged culture. Moreover, veratrole in high concentrations (> 0.1% w/v), unlike diphenylamine, inhibits the formation of phytoene, resulting in an almost total block of carotenoid biosynthesis.     

Flowthrough Reactor Flasks for Study of Microbial Metabolism in Sediments

Flowthrough reactor flasks are described that allow continuous low-level nutrient input to mixed anoxic sediments without dilution of the sediment. The flasks were tested by simulating sulfate inputs into sediments collected from a freshwater eutrophic lake. After an initial 2-day adaptation within the reactor system, rates of methane production and sulfate consumption were constant for the duration of a 12-day incubation. A sulfate input rate of 0.15 mmol liter of sediment⁻¹ day⁻¹ resulted in an equivalent rate of sulfate removal, which was unaffected by inputs of acetate (1.0 mmol liter of sediment⁻¹ day⁻¹). The rate of methane production in control reactors, 0.18 mmol liter of sediment⁻¹ day⁻¹, was doubled by the addition of acetate, whereas sulfate consumption was only stimulated by additions of high concentrations of sulfate plus acetate (1.5 and 1.0 mmol liter of sediment⁻¹ day⁻¹, respectively). The reactor system appears to be effective in maintaining the balance between sulfate reduction and methane production in freshwater sediments and is potentially useful for study of the response of sediment populations to varying inputs of naturally occurring substrates, selected inhibitors, or xenobiotic compounds.     

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     

Protease and carotenogenesis in Blakeslea trispora

Carotene production by single and mated Blakeslea trispora has been studied. On mating and on the addition of trisporic acid to minus cultures there was an increase in the membrane bound neutral protease (MW 126 000) activity. The protease probably acts by inactivating the inhibitory protein of carotene biosynthesis resulting in increased carotenogenesis.     

Enhancement of carotenoid synthesis by fungal metabolites

Carotenoids, mainly the all-trans--carotene, may be produced by the fungus Phycomyces blakesleeanus under normal fermentation conditions. A number of chemical compounds that stimulate or inhibit fungal carotenogenesis have been reported earlier. A procedure for the screening of organisms that enhance carotenogenesis on co-culturing with P. blakesleeanus is described. A water-soluble stimulator of carotenogenesis was obtained from the fermentation broth of an unidentified Aspergillus sp. isolated from soil. The crude preparation appears to be ten times more potent than dimethylphthalate.     

Sexualreaktion und Carotin-Synthese bei Zygomyceten

Zusammenfassung Gamonbildung und Carotinsynthese sind bei den Zygomyceten Mucor mucedo, Blakeslea trispora und Phycomyces Blakesleeanus gekoppelte Reaktionen. Die Gamonbildung verläuft parallel zur Carotinbiosynthese. Damit kann ein Weg zur Klärung der Gamonsynthese gefunden werden.

---

Sexual reaction and carotene synthesis in Zygomycetes

Summary In the Zygomycetes Mucor mucedo, Blakeslea trispora and Phycomyces Blakesleeanus, gamon formation and carotene synthesis are coupled reactions. The gamon formation goes parallel with the carotene biosynthesis. This provides a way by which gamon synthesis can be elucidated.

     

ENVIRONMENTAL FACTORS AND SEXUAL EXPRESSION IN CHLOROCOCCUM ECHINOZYGOTUM (CHLOROPHYCEAE)1

Several environmental factors affected total growth and zygospore production in Chlorococcum echinozygotum Starr. The temperature range at which zygospore production occurred was more restricted than the range that supported vegetative growth. Light intensity had little effect upon zygospore formation: gamete production and gamete pairing occurred in darkness. Zygospore production occurred over a wide pH range; bicarbonate had a minor effect upon zygospore formation. Nitrogen concentration was the factor of primary importance. As the level of nitrogen supplied as nitrate, ammonia, urea and asparagine in the medium was increased, zygospore production first increased (over no nitrogen) at low levels and then decreased at high levels. All levels of glutamine supplied reduced zygospore production. A possible way in which nitrogen concentration in the medium and sexual expression are linked is discussed.     

New Mutants of Phycomyces blakesleeanus for (beta)-Carotene Production

The accumulation of (beta)-carotene by the zygomycete Phycomyces blakesleeanus is increased by mutations in the carS gene. The treatment of spores of carS mutants with N-methyl-N(prm1)-nitro-N-nitrosoguanidine led to the isolation, at very low frequencies, of mutants that produced higher levels of (beta)-carotene. Strain S556 produced about 9 mg of (beta)-carotene per g of dry mass when it was grown on minimal agar. Crosses involving strain S556 separated the original carS mutation from a new, unlinked mutation, carF. The carF segregants produced approximately as much carotene as did carS mutants, but they were unique in their ability to produce zygospores on mating and in their response to agents that increase carotenogenesis in the wild type. The carotene contents of carF segregants and carF carS double mutants were increased by sexual interaction and by dimethyl phthalate but were not increased by light or retinol. Mixed opposite-sex cultures of carF carS mutants contained up to 33 mg of (beta)-carotene per g of dry mass. Another strain, S444, produced more (beta)-carotene than did S556 but was marred by slow growth, defective morphology, and bizarre genetic behavior. In all the strains tested, the carotene concentration was minimal during the early growth phase and became higher and constant for several days in older mycelia.     

Modeling of Growth of Lactobacillus sanfranciscensis and Candida milleri in Response to Process Parameters of Sourdough Fermentation

We investigated the effect of the ecological factors pH, temperature, ionic strength, and lactate, acetate, and ethanol levels on Candida milleri and two strains of Lactobacillus sanfranciscensis, organisms representative of the microflora of sourdough. A mathematical model describing the single and combined effects of these factors on the growth of these organisms was established in accordance with the following criteria: quality of fit, biological significance of the parameters, and applicability of the in vitro data to in situ processes. The growth rates of L. sanfranciscensis LTH1729 and LTH2581 were virtually identical under all conditions tested. These organisms tolerated >160 mmol of undissociated acetic acid per liter. Growth occurred in the pH range of 3.9 to 6.7 and was completely inhibited by 4% NaCl. C. milleri had a lower optimum temperature for growth (27°C) than the lactobacilli. The growth of the yeast was not affected by pH in the range of 3.5 to 7, and up to 8% NaCl was tolerated. Complete inhibition of growth occurred at 150 mmol of undissociated acetic acid per liter, but acetate at concentrations of up to 250 mmol/liter exerted virtually no effect. The model provides insight into factors contributing to the stability of the sourdough microflora and can facilitate the design of novel sourdough processes.     

Effects of chemical components and nitrogen sources on zygospore development inPhycomyces blakesleeanus

We examined the effects of chemical components and nitrogen sources on zygospore development, using 62 different ingredients based on Sutter's synthetic medium Sl, which has been widely used for studies of sexual physiology inPhycomyces. An increase of inorganic microelements such as ZnSO₄, NaMoO₄ and CaCl₂ promoted an increase in the number of zygospores per unit area. Glutamate (Glu) contained in Sl as the sole nitrogen source was indispensable for sexual development, and replacement of Glu with NH₄ ⁺ (Am) strongly inhibited it, mainly because of growth inhibition. However, zygospore production was enhanced 1.8-fold by equivalent amounts of both Glu and Am as compared with Glu alone. A newly developed medium, mSl+Am, enriched with Am and the above-mentioned effective microelements doubled the number of zygospores formed per unit area (density), compared with Sutter's original Sl, and increased both the density and the weight (volume) of zygospores, 1.6- and 2-fold, respectively, compared with potato-dextrose-agar medium enriched with yeast extract and casitone (PDAYC). Sexual stimulation by mSl+Am was also observed in the mating of a pair of β-carotene-deficient mutants. Methionine sulfoxime, an inhibitor of glutamine synthetase, strongly inhibited the progress of mating without significant growth inhibition. The first and the second authors contributed equally to this work.     

Spore activation by acetate, propionate and heat in Phycomyces mutants

Summary Exposure to heat, acetate, or propionate activates the spores of the fungus Phycomyces blakesleeanus and allows them to germinate. Using counterselection with the antibiotic N-glycosyl-polyfungin, seven mutants were isolated on the basis of decreased spore activation by acetate and two on the basis of decreased spore activation by propionate. The nine mutants showed decreased activation by both chemicals and by heat, increased heat lethality, and altered patterns of trehalase activation. These and other observations indicate that spore activation by the three agents and spore death by heat are mediated by the same cellular component(s), which is probably involved in the regulation of cyclic AMP concentration.