Ubiquinone and carotene production in the <Emphasis Type="Italic">Mucorales Blakeslea</Emphasis> and <Emphasis Type="Italic">Phycomyces</Emphasis>

The filamentous fungi Phycomyces blakesleeanus and Blakeslea trispora (Zygomycota, Mucorales) are actual or potential industrial sources of β-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.     

Zygote Formation in Blakeslea trispora: Morphological Peculiarities and Relationship with Carotenoid Synthesis

Changes associated with zygospore formation in the mucorous fungus Blakeslea trispora were studied. Zygospores are dormant cells with thickened cell walls and large central lipid vacuoles containing large amounts of lycopene. We established for the first time that B. trispora gametangia of different sexes differ in their carotenoid content and revealed that zygote formation involves a novel structure that consists of densely intertwined hyphae. Using inhibitory analysis (blocking -carotene synthesis with diphenylamine and 2-amino-6-methylpyridine), we showed that suppression of carotene producion results in the inhibition of zygote formation. Hence, we established a manifest dependence of zygote formation on -carotene synthesis.     

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 β-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), β-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.     

Effects of some non-pathogenic micro-organisms on the growth of gnotobiotic barley plants

Summary Mated cultures of Blakeslea trispora, which produce trisporic acid, inhibited root and shoot extension of barley plants grown in gnotobiotic cultures, where the carbon from roots was the sole source of carbon available to the microorganism. Unmated cultures of B. trispora and Mucor hiemalis, which do not synthesize trisporic acid, inhibited root extension only. Azotobacter chroococcum had a variable effect in different experiments on both root and shoot extension but could at least partially relieve the inhibitory effect of B. trispora on root growth. Plant growth regulators, applied to roots exogenously, produced effects on plants which, although comparable with some of the inhibitory effects of the micro-organisms, were not comparable with the stimulatory effects of A. chroococcum. re]19751127     

Untersuchungen zur Aktivierung von Sporenhomogenaten durch Wärmebehandlung

Zusammenfassung Homogenate hergestellt aus ruhenden Sporen von Phycomyces blakesleeanus lassen sich durch eine Wärmebehandlung aktivieren, d. h. sie produzieren im Vergleich zur nicht-wärmeaktivierten Homogenat-Kontrolle verstärkt Lactat, Äthanol, Pyruvat und Acetaldehyd. Die höchsten Werte liefert dabei eine Wärmebehandlung von 4 min bei 56°C.Der Zusatz von d-Glucose und d-Fructose-1,6-diphosphat führt sowohl bei wärmebehandelten wie nicht-wärmebehandelten Sporenhomogenaten im Vergleich zur entsprechenden Kontrolle nicht zu einem erhöhten Anstau der vier untersuchten Glykolyseprodukte. d-Glycerat-2-phosphat bzw. d-Glycerat-3-phosphat werden dagegen von einem unvorbehandelten Homogenat sehr gut umgesetzt; die Leistung eines wärmeaktivierten Homogenats ist unter entsprechenden Versuchsbedingungen nur geringfügig höher. d-Glycerinaldehyd-3-phosphat wird dagegen von einem wärmeaktivierten Homogenat weitaus besser umgesetzt als von der nicht-wärmebehandelten Kontrolle.

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Heat-activation of cell-free extracts from dormant spores of Phycomyces blakesleeanus. VII

Summary Cell-free extracts from dormant spores of Phycomyces blakesleeanus can be activated by a heat shock, that means they show an enhanced level of L-lactate, ethanol, pyruvate and acetaldehyde compared to the untreated control.Adding of d-glucose and d-fructose-1,6-diphosphate does not lead to an enhanced level of the four metabolic intermediates of glycolysis by activated and not activated cell-free extracts as well in respect of the corresponding control.The turnover of 3-P-d-glycerate and 2-P-d-glycerate, however, is higher even by the untreated extract; the capacity of a heat shocked extract is insignificantly higher.The turnover of d-glyceraldehyde-3-phosphate is remarkably better by an activated cell-free extract with regard to the not activated control.

     

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.     

Biotoxic activity in the Mucorales

The toxigenicity of representatives of 15 species of Mucorales (Absidia glauca, Actinomucor elegans, Cunninghamella elegans, Helicostylum piriforme, Mortierella isabellina, Mortierella (Mucor) rammaniana, Mucor hiemalis, Mucor mucedo, Mucor spinosus, Phycomyces blakesleeanus, Rhizopus oligosporus, Rhizopus stolonifer, Syncephalastrum racemosum, Thamnidium elegans, Zygorhynchus moelleri) towards the larvae of brine shrimp (Artemia salina) and the growth of pea seedlings (Pisum sativum) and tobacco plants (Nicotiana tabacum) was evaluated. The fungi were cultivated on malt extract agar and aqueous solutions of the cultures were tested.Thamnidium elegans showed a marked toxic action towards brine shrimp (mortality: 74.1%) andPhycomyces, Actinomucor andSyncephalastrum were only weakly toxic. Length and weight of stems of pea seedlings were moderately reduced by extracts ofAbsidia, Cunninghamella, Zygorhynchus andThamnidium and to a lesser degree byMucor spinosus. Cunninghamella andMucor spinosus also inhibited the development of pea hypocotyls. The length of tobacco stems was reduced byMortierella ramanniana, Rhizopus stolonifer andCunninghamella elegans. Wilting or other toxic phenomena were never observed with both test plants. Considering the present results and data from literature it is suggested that species of Mucorales have only a weak toxigenicity.     

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.     

Stimulation of Carotenogenesis by Microbial Cells

Spent mycelia of Blakeslea trispora recovered from a previous fermentation enhanced carotene production by mated cultures of the same organism. Peak yields of 107 to 142 mg of carotene per 100 ml of medium were achieved in 6 days. β-Carotene constituted 92% of the total carotenoids produced. The enhancing substance was found present in an aqueous extract of the mycelium. Part of its activity was due to an organic acid fraction. Other microbial cells, molds, yeasts, and bacteria were also capable of enhancing carotene production.     

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.     

Effect of the ratio of (+) and (−) mating type of Blakeslea trispora on carotene production from cheese whey in submerged fermentation

The effect of the ratio of (+) and (?) mating type of Blakeslea trispora on carotene production from deproteinized hydrolysed whey in shake flask culture was investigated. Also, the inoculum ratio of the two mating types on the morphology of the microorganism and the relationship between morphological changes of the fungus and product formation were studied. The concentration of carotenes was significantly affected by the ratio of (+) and (?) mating type of B. trispora. A ratio of 1:10 up to 1:100 of (+) and (?) type was found to achieve the highest carotene yields. The optimum ratio of the (+) and (?) mating types for the maximum pigment production (175.0 mg/g dry biomass at 8 days of fermentation) was found to be 1:10. The carotene content in B. trispora consisted of β-carotene, γ-carotene, and lycopene. At the maximum concentration of carotenes the proportion of β-carotene, γ-carotene, and lycopene (as percent of total carotenes) was 80, 12, and 8%, respectively. B.trispora growing in submerged fermentation is able to develop complex morphologies which have been classified into three major groups: freely dispersed hyphae, clumps and pellets. These parameters are strongly influence the production of carotenes.     

Synthese von Speicherstoffen aus Pyruvat durch Rhodospirillum rubrum

Zusammenfassung Pyruvat kann durch belichtete Zellen von R. rubrum assimiliert und zur Synthese verschiedener Zellbestandteile verwendet werden. Ist das Angebot an Substrat und Lichtenergie groß genug, so wird ein Teil der Brenztraubensäure in Speicherstoffe eingebaut. Suspension der Zellen in Phosphat-puffer und Inkubation unter Wasserstoff stimuliert die Bildung des Speicherstoffes Poly--Hydroxybuttersäure. Die Synthese eines ebenfalls als Speicherstoff in R. rubrum bekannten Polysaccharids konnte vor allem bei großem Substratüberfluß und Inkubation unter Stickstoff beobachtet werden; es wurde jedoch auch unter optimalen Bedingungen nur ein geringer Anteil der total aufgenommenen Brenztraubensäure zur Synthese dieses Speicherpolysaccharids verwendet.

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Synthesis of storage material from pyruvate by Rhodospirillum rubrum

Summary Illuminated cells of R. rubrum assimilate pyruvate and use it for the ynthesis of different cell components. A sufficient supply of substrate and light energy provided, part of the pyruvate is built into storage products. If the cells are suspended in phosphate buffer and kept under an atmosphere of molecular hydrogen the formation of the -hydroxybutyrate polymer is stimulated. An excess of substrate and incubation under molecular nitrogen leads to the synthesis ofa polysaccharide, also known as storage product in R. rubrum; but even under optimal conditions only a small part of the pyruvate is used for the synthesis of this storage polysaccharide.

     

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.     

Mycoflora of the Finnish “sauna” (bath-house)

The investigation concerned the mycoflora of the old, in some cases already discarded, bath houses (sauna) situated in various parts of Finland for the purpose of finding the fungus,Phialophora pedrosoi, which causes chromoblastomycosis in man. The material, 220 specimens, consisting of thin shavings from the wooden floors and platforms, was collected from 78 bath houses. P. pedrosoi was not found in this investigation; the followingPhialophora-species:P. fastigiata, P. lagerbergii, P. lignicola andP. verrucosa were isolated, however.In total 98 various fungus species were isolated, the number of isolates was 1052. Phycomycetes represented 9.2 % of the species and 15.2 % of the isolates, Ascomycetes 4.1 % and 3.4 %, Basidiomycetes 4.1 % and 0.7 % and Fungi imperfecti 82.6 % and 80.7 % respectively of the species and the isolates. The species most frequently isolated from specimens were:Trichoderma viride, Mucor mucedo, Stemphylium consortiale, Rhinocladiella atrovirens, Arthrobotrys superba andPhialophora verrucosa.In all, 13 nematode-destroying fungus species were isolated belonging to the genera:Nematoctonus, Cephalosporium, Harposporium, Dactylaria, Arthrobotrys andDactylella. 60 % of the bath houses were infected by nematodes. Gliodendron gen. nov. with the speciesGliodendron balnicolum sp. nov. is described and illustrated.

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Zusammenfassung Die Untersuchung betraf die Mykoflora von alten, in manchen Fällen bereits aufgegebenen Badehäusern, die sich in verschiedenen Teilen Finnlands befanden, um den Pilz,Phialophora pedrosoi, den Erreger der Chromoblastomykose, zu finden. Das Material bestand aus 220 Einzelstücken, Spänen von Holzfußböden, die in 78 Badehäusern gesammelt wurden,Phialophora pedrosoi wurde in diesen Untersuchungen nicht gefunden. Die folgendenPhialophora-Arten sind isoliert worden:P. fastigiata, P. lagerbergii, P. lignicola, undP. verrucosa. Insgesamt sind 98 verschiedene Pilzarten isoliert worden. Die Anzahl der Isolierungen war 1052. Phykomyceten stellten 9,2 % von den Arten und 15,2 % der Isolierungen dar: Askomyceten 4,1 % und 3,4 %; Basidiomyceten 4,1 % und 0,7 %; Fungi imperfecti 82,6 % und 80,7 %. Die häufigst isolierten Arten waren:Trichoderma viride, Mucor mucedo, Stemphylium consortiale, Rhinocladiella atrovirens, Arthrobotrys superba undPhialophora verrucosa. Alles in allem wurden 13 Nematoden zerstörende Arten isoliert, die zu den folgenden Gattungen gehörten:Nematoctonus, Cephalosporium, Harposporium, Dactylaria, Arthrobotrys undDactylella. Sechzig Prozent der Badehäuser waren von Nematoden infektiert.Gliodendron gen. nov. ist mit der ArtGliodendron balnicolum, sp. nov. beschrieben und abgebildet worden.

     

Relationship Between Sexuality and Carotene Synthesis in Blakeslea trispora

When stimulated by equivalent amounts of progametangia-inducing hormones, cultures of the minus mating type of Blakeslea trispora produce about the same quantities of carotenoids as mated cultures of the fungus, which suggests that the stimulation of carotene synthesis during the sexual activity of mated cultures is the result of hormonal action. These hormones were isolated and purified. From spectroscopic analysis of purified samples, it appears that the hormones are identical with trisporic acids B and C. When both mating types of B. trispora were cultivated in one vessel but were kept apart by membrane filters, the formation of sex hormones was not inhibited. Physical contact between the mating types is obviously not required for the induction of sexual activity. The sex hormones also formed in combined cultures of B. trispora-plus and Zygorhynchus moelleri (a homothallic species), but not in combined cultures of B. trispora-minus and Z. moelleri. This is evidence for the hypothesis that the hormones are produced by B. trispora-plus only.     

Ultrastruktur und polysaccharidanteile der cuticula von Triops cancriformis Bosc. (Crustacea,Notostraca) während der Häutungsvorbereitung

Zusammenfassung Die Cuticula von Triops cancriformis besteht aus Endo-, Exo- und Epicuticula. Die Epicuticula ist aus vier Schichten aufgebaut, die Exocuticula aus 10 und die Endocuticula aus 60–80. Die Schichten der Endocuticula sind aus annähernd oberflächenparallel verlaufenden Mikrofibrillen, die zu Lamellulae zusammentreten, gebildet. Diese Lamellulae atehen senkrecht zur Oberfläche. Die Lamellulae der einzelnen Lagen verlaufen im rechten Winkel zu denen der Nachbarlagen. In Sinnesborsten verläuft so ein Teil der Fibrillen in Längsrichtung, der andere quer zur Längsachse.Polysaccharide finden sich in den Lamellulae, in zwei Schichten der Epicuticula, den Desmosomen und als Glykogengranula in Epidermiszellen.Die Häutung zeigt anscheinend keine Besonderheiten gegenüber anderen Arthropoden.

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Ultrastructure and polysaccharide content of the cuticle of Triops cancriformis Bosc. (Crustacea, Notostraca) during the Molting preparation

Summary The cuticle of Triops cancriformis consists of endo-, exo- and epicuticle. The epicuticle comprises 4 layers, the epocuticle 10 and the endocuticle 60–80 layers. The layers of the endocuticle consist of microfibrils. These microfibrils are almost parallel to the surface of the cuticle and merge into lamellulae. These lamellulae run vertical to the surface. The lamellulae in any one layer are at right angles to the lamellulae in the neighbouring layers. Thus, some of the fibrils in sensory setae are parallel to the longitudinal axis and others are perpendicular to it.Polysaccharides are found in the lamellulae, in two layers of the epicuticle, in the attachment regions and in the glycogen deposits in the epidermis cells.The molting process seems to be similar to the molting process of other arthropoda.

     

Carotene production from waste cooking oil by Blakeslea trispora in a bubble column reactor: The role of oxidative stress

The oxidative stress induced by hydroperoxides and reactive oxygen species (ROS) during carotene production from waste cooking oil (WCO) and corn steep liquor (CSL) by the fungus Blakeslea trispora in a bubble column reactor was investigated. The specific activities of the intracellular enzymes superoxide dismutase (SOD) and catalase (CAT) as well as the micromorphology of the fungus were measured in order to study the response of the fungus to oxidative stress. The changes of the morphology of microorganism leaded to pellets formation and documented using a computerized image analysis system. As a consequence of the mild oxidative stress induced by hydroperoxides of WCO and ROS a significant increase in carotene production was obtained. The highest carotene concentration (980.0 mg/l or 51.5 mg/g dry biomass) was achieved in a medium consisted of CSL (80.0 g/L) and WCO (50.0 g/L) at an aeration rate of 5 vvm after 6 days of fermentation. In this case the carotenes produced consisted of β‐carotene (71%), γ‐carotene (26%), and lycopene (3%). The strong oxidative stress in the fungus caused a significant increase of γ‐carotene concentration. Bubble column reactor is a useful fermentation system for carotene production in industrial scale.     

Beziehungen zwischen Proteinsynthese und Alkaloidbildung beim Mutterkornpilz in saprophytischer Kultur

Zusammenfassung Zur Klärung der Frage, ob und in welchem Maße bei demClaviceps-StammSD 58 in der stationären Wachstumsphase, in der die Alkaloid-bildung vor sich geht, eine Proteinsynthese erfolgt, wurde die Inkorporationsrate von³⁵S-Sulfat und³⁵S-Methionin in die Proteinfraktion in Abhängigkeit vom Alter der Kultur untersucht. Die Proteinsynthese geht auch nach Abschluß des Wachstums, wenn der Gehalt an Protein nicht mehr zunimmt, weiter. Das Ausmaß beträgt je nach eingesetztem Tracer 10–50% der während des Wachstums erfolgenden Synthese. Der übergang von der Wachstums- zur stationären Phase ist durch einen erneuten Anstieg der Proteinsynthese gekennzeichnet. Die nach Abschluß des Wachstums fortlaufende Synthese und der damit verbundence Turnover lassen es möglich erscheinen, daß die für die Alkaloidbildung verantwortlichen Enzyme neu gebildet werden und daß die Aminosäurebausteine der Alkaloide aus dem Eiweißabbau stammen.

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Relations between the syntheses of protein and alkaloids inClaviceps

Summary InClaviceps strainSD 58 the formation of alkaloids takes place mainly in the stationary phase of growth. In order to determine the rate of protein synthesis in this phase, the incorporation of³⁵S labeled sulfate and methionine was investigated. When sulfate is applied the rate of protein synthesis in the stationary phase is 10% of that of the growth phase and 50% when methionine is used. In the decreasing growth phase there is a second smaller maximum of protein synthesis. The function of protein synthesis in the stationary growth phase with respect to the formation of the enzymes of alkaloid biosynthesis is discussed. In addition the role of protein turnover as a source of amino acids for the alkaloid biosynthesis is considered.