Effect of mutation on synthesis of alkaloid byPenicillium roquefortii VKM F-141 andP. fellutanum VKM F-1073

Mutant strains ofPenicillium roquefortii VKM F-141 andP. fellutanum VKM F-1073 were obtained by mutagenesis induced by ultraviolet irradiation,N-methyl-N-nitronitrosoguanidine, and bromouracil. By the rates of alkaloid production, the mutant strains can be divided into three groups: (1) unable to synthesize alkaloids; (2) with a high rate of biosynthesis; (3) with changed alkaloid composition. Compounds not characteristic of wild-type cultures were found in alkaloid fractions of some mutant strains.     

Production of mycophenolic acid by fungi of the genus Penicillium link

Out of 36 strains of fungi of the genus Penicillium, some of which were isolated from ancient permafrost soils, 14 strains synthesized mycophenolic acid (MPA). Maximal (over 500 mg/l) accumulation of MPA in culture liquid was observed in P. brevicompactum strains (VKM F-457, VKM F-477, and VKM F-1150). This was the first study to detect MPA in representatives of the species P. rugulosum; in three strains of this species (VKM FW-665, VKM FW-717, and VKM FW-733), the level of MPA accumulation exceeded 300 mg/l. The time course of the synthesis of MPA by the P. rugulosum strain VKM FW-733 was studied. It was shown that the synthesis of this metabolite was dramatically intensified at the stationary growth phase (ten days).     

The Alkaloids of Penicillium aurantiogriseum Dierckx (1901) var. aurantiogriseum VKM F-1298

The fungus Penicillium aurantiogriseum var. aurantiogriseum VKM F-1298 produces two benzodiazepine alkaloids (anacine and aurantine) and one diketopiperazine alkaloid (aurantiamine). When cultured in a submerged mode in Abe medium, the alkaloids are mostly secreted into the medium. The dynamics of aurantine and aurantiamine accumulation in the medium is characterized by the presence of a relatively sharp maximum in the idiophase, whereas the accumulation of anacine in the medium is characterized by an extended plateau and occurs concurrently with fungal growth.     

Formation of alkaloids from Penicillium species fungi during growth on wheat kernels

The ability to produce alkaloids has been studied in 13 strains belonging to 10 species of the genus Penicillium. Most of these strains produce identical ranges of alkaloids when grown on wheat grain and synthetic Abe medium. They are roquefortine, 3,12-dihydroroquefortine, and glandicolines A and B in strain P. chrysogenum VKM F-1987; fumigaclavines A and B, festuclavine, and pyroclasine in P. commune VKM F-308, F-3491, and KBP4; agroclavine 1 and epoxyagroclavine 1 in P. fellutanum VKM F-1073; fellutanine A in P. fellutanum F-3020; roquefortine, 3,12-dihydroroquefortine, meleagrin, and glandicolines A and B in P. glandicola VKM F-743; aurantioclavine in P. nalgiovense VKM F-229; isofumigaclavines A and B, festuclavine, roquefortine, and 3,12-dihydroroquefortine in P. roquefortii VKM F-2389; roquefortine, 3,12-dihydroroquefortine, and meleagrin in P. vitale VKM F-3624; roquefortine and oxaline in P. vulpinum VKM F-256; and alpha-cyclopiazonic acid and rugulovasine B in P. viridicatum C-47. No alkaloids were found in P. rugulosum VKM F-352 grown on wheat grain. A simple method is proposed for isolating alkaloids from affected grains.     

The alkaloids of Penicillium aurantiogriseum Dierckx (1901) var. aurantiogriseum VKM F-1298

The fungus Penicillium aurantiogriseum var. aurantiogriseum VKM F-1298 produces two benzodiazepine alkaloids (anacine and aurantine) and one diketopiperazine alkaloid (aurantiamine). When cultured in a submerged mode in Abe medium, the alkaloids are mostly secreted into the medium. The dynamics of aurantine and aurantiamine accumulation in the medium is characterized by the presence of a relatively sharp maximum in the idiophase, whereas the accumulation of anacine in the medium is characterized by an extended plateau and occurs concurrently with fungal growth.     

Production of mycophenolic acid by fungi of the genus <Emphasis Type="Italic">Penicillium</Emphasis> link

Out of 36 strains of fungi of the genus Penicillium, some of which were isolated from ancient permafrost, 14 strains synthesized mycophenolic acid (MPA). Maximal (over 500 mg/l) accumulation of MPA in culture liquid was observed in P. brevicompactum strains (VKM F-457, VKM F-477, and VKM F-1150). This was the first study to detect MPA in representatives of the species P. rugulosum; in three strains of this species (VKM FW-665, VKM FW-717, and VKM FW-733), the level of MPA accumulation exceeded 300 mg/l. The time course of the synthesis of MPA by P. rugulosum strain VKM FW-733 was studied. It was shown that the synthesis of this metabolite was dramatically intensified during the stationary growth phase (ten days).Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 1, 2005, pp. 95–98.Original Russian Text Copyright © 2005 by Vinokurova, Ivanushkina, Kochkina, Arinbasarov, Ozerskaya.     

Effect of microelements on the biosynthesis of secondary metabolites by the fungusPenicillium citrinum thom VKM F-1079

Penicillium citrinum VKM F-1079 was found to produce clavine ergot alkaloids and citrinin, a secondaryO-heterocyclic metabolite. Citrinin was produced in the idiophase, whereas the production of ergot alkaloids paralleled fungal growth. The addition of manganese ions to the growth medium stimulated the biosynthesis of both citrinin and ergot alkaloids. Zinc ions stimulated only citrinin synthesis. The presence of these microelements in the growth medium influenced the proportion between the ergot alkaloids synthesized. Copper, manganese, and iron ions slightly affected fungal growth and alkaloid production. The effect of microelements on the main kinetic parameters of growth and alkaloid production was studied.     

Novel laccase—producing ascomycetes

Screening of ascomycetes producing laccases during growth on agar medium or submerged cultivation in the presence of various natural sources of carbon and energy (grain crops and potato) was carried out. The conditions of submerged cultivation of the most active strains (Myrothecium roridum VKM F-3565, Stachybotrys cylindrospora VKM F-3049, and Ulocladium atrum VKM F-4302) were optimized for the purpose of increasing laccase activity. The pH-optima and substrate selectivity of laccases in the culture liquid of the strains in relation to ABTS and phenolic compounds (2,6-dimethoxyphenol, syringaldazine, ferulic acid, p-coumaryl alcohol, and coniferyl alcohol) were investigated. High laccase activity at neutral pH was shown for the culture liquids of M. roridum VKM F-3565 and S. cylindrospora VKM F-3049 strains that provides prospects for using laccases of these strains in various cell biotechnologies.     

The Penicillium commune Thom and Penicillium clavigerum Demelius Fungi Producing Fumigaclavines A and B

The type strains Penicillium clavigerum VKM F-447 and P. commune VKM F-3233 are found to produce fumigaclavines A and B. Of the seven other strains of these species, only two strains, P. commune VKM F-3088 and F-3491, possess the ability to synthesize these alkaloids. It is suggested that the five other strains under study either lost such an ability or require very specific conditions for the synthesis of these alkaloids.     

Production of Alkaloids by Fungi of the Genus PenicilliumGrown on Wheat Grain

The ability to produce alkaloids has been studied in 13 strains belonging to ten species of the genus Penicillium. Most of these strains produce identical ranges of alkaloids when grown on wheat grain and synthetic Abe's medium. These are roquefortine, 3,12-dihydroroquefortine, and glandicolines A and B in strain P. chrysogenum VKM F-1987; fumigaclavines A and B, festuclavine, and pyroclavine in P. commune VKM F-308, F-3491, and KBP4; agroclavine 1 and epoxyagroclavine 1 in P. fellutanum VKM F-1073; fellutanine A in P. fellutanum F-3020; roquefortine, 3,12-dihydroroquefortine, meleagrin, and glandicolines A and B in P. glandicola VKM F-743; aurantioclavine in P. nalgiovense VKM F-229; isofumigaclavines A and B, festuclavine, roquefortine, and 3,12-dihydroroquefortine in P. roquefortii VKM F-2389; roquefortine, 3,12-dihydroroquefortine, and meleagrin in P. vitale VKM F-3624; roquefortine and oxaline in P. vulpinum VKM F-256; and -cyclopiazonic acid and rugulovasine B in P. viridicatum C-47. No alkaloids were found in P. rugulosum VKM F-352 grown on wheat grain. A simple method is proposed for isolating alkaloids from affected grain.     

Mechanisms of the alkaloid aurantioclavine excretion and uptake during Penicillium nalgiovense VKM F-229 growth

The biphasic dynamics of the alkaloid aurantioclavine in the culture liquid of Penicillium nalgiovense VKM F-229 is shown to be due to the diauxic growth of the fungus on two carbon sources, succinate and mannitol. In the phase of active growth on succinate, the fungus synthesizes aurantioclavine and excretes it into the medium in an energy-independent manner, as a result of which the concentration of the alkaloid in the culture liquid rises. During the phase of metabolic adaptation to the other carbon source, mannitol, the concentration of aurantioclavine in the culture liquid falls, probably due to the energy-dependent uptake of the alkaloid by fungal cells. The reversible excretion of aurantioclavine in P. nalgiovense indicates that these are regulated processes and depend on the growth parameters and the physiological state of the fungus.     

Germination of spores of mycelial fungi in relation to exogenous dormancy

Comparative analysis of germination of asexual sporulation spores (conidia and sporangiospores) and of specific features of dormancy release was carried out for ascomycete mycelial fungi Aspergillus tamarii VKM F-64 and A. sydowii VKM F-441, as well as for zygomycete fungi Cunninghamella echinulata VKM F-663 and Umbelopsis ramanniana VKM F-582. The spores of these strains were shown to be in a state of exogenous dormancy and differed in lag phase duration and germination rate, which depended on the presence of nutrients in the medium. Only the strain C. echinulata VKM F-663 exhibited 100% spore germination, with the germination rate and lag phase duration not depending on the composition of the medium. While in A. tamarii strain VKM F-64, the total number of spores germinating on rich and poor media was also almost the same, in the absence of nutrients lag phase duration increased and the germination rate decreased. For strains U. ramanniana VKM F-582 and A. sydowii VKM F-441, the degree of spore germination in the absence of nutrients in the medium was considerably lower than on the rich medium, while the lag phase was longer. These data indicate that the spores of C. echinulata VKM F-663 are in the state of exogenous dormancy, which does not require for release any compounds except water. The spores of U. ramanniana strain VKM F-582 and of the Aspergillus strains exhibited another variant of exogenous dormancy, which required for release, apart from water, also the sources of carbon and nitrogen. Thus, the character of dormancy release may differ even within a single genus (Aspergillus).     

Selection of Ergot Alkaloid Producers by Induced Mutagenesis

Using the induced mutagenesis technique, a series of genetically modified Clavicepssp. VKM F-2609 strains that display high levels of agroclavine and elymoclavine synthesis were selected by induced mutagenesis. Compared to the parent strain, c106 displayed a 40-fold higher level of agroclavine synthesis, and c66 displayed an eightfold higher level of elymoclavine synthesis. The levels of synthesis of other alkaloids were decreased in these strains. The effects of various carbohydrates on the strain growth and ergot alkaloid biosynthesis was then investigated in both the parent strain and c106. The largest amount of agroclavine was synthesized by c106 strain growing on a medium with maltose.     

Lipid synthesis by <Emphasis Type="Italic">Geomyces pannorum</Emphasis> under the impact of stress factors

Lipogenic activity and fatty acid composition of two strains of Geomyces pannorum were studied in the course of fungal growth. The strains were isolated from an Arctic cryopeg lens (VKM FW-2241) and from Central Russia (VKM F-3808). The adaptive reactions in both strains towards the temperature decreasing to 2°C involved intensification of the fatty acid desaturation. The degree of lipid unsaturation increased mainly due to a higher amount of α-linolenic acid (α-C_18:3) especially in the case of strain VKM FW-2241. Elevated NaCl concentration in the medium enhanced the level of linoleic acid (C_18:2) which apparently played a specific role in osmoprotection. Strain VKM FW-2241 was more tolerant to increased salinity than strain VKM F-3808. Almost complete inhibition of the growth of strains VKM F-3808 and VKM FW-2241 occurred at salinity of 10 and 20%, respectively; however, the viability of the strains was not affected. Under the combined effect of high salinity and hypothermia, the ratio between C_18:2 and α-C_18:3 acids was intermediate, indicating that these acids were involved in two adaptation mechanisms. The inhibition of fungal growth under stress was found to result in lipid overproduction. An increased pool of energy-rich lipids in fungi possibly contributes to their strategy of cell survival.     

Effect on microelements on biosynthes of secondary metabolites in the fungus Penicillium citrinum Thom VKM F-1079

Penicillium citrinum VKM F-1079 was found to produce clavine ergot alkaloids and citrinin, a secondary O-heterocyclic metabolite. Citrinin was produced in the idiophase, whereas the production of ergot alkaloids paralleled fungal growth. The addition of manganese ions to the growth medium stimulated the biosynthesis of both citrinin and ergot alkaloids. Zinc ions stimulated only citrinin synthesis. The presence of these microelements in the growth medium influenced the proportion between the ergot alkaloids synthesized. Copper, manganese, and iron ions affected but little fungal growth and alkaloid production. The effect of microelements on the main kinetic parameters of growth and alkaloid production was studied.     

On the Mechanisms of the Excretion and Uptake of the Alkaloid Aurantioclavine during the Growth of the Fungus Penicillium nalgiovense VKM F-229

The biphasic dynamics of the alkaloid aurantioclavine in the culture liquid of P. nalgiovense VKM F-229 is shown to be due to the diauxic growth of the fungus on two carbon sources, succinate and mannitol. In the phase of active growth on succinate, the fungus synthesizes aurantioclavine and excretes it into the medium in an energy-independent manner, as a result of which the concentration of the alkaloid in the culture liquid rises. During the phase of metabolic adaptation to the other carbon source, mannitol, the concentration of aurantioclavine in the culture liquid falls, probably due to the energy-dependent uptake of the alkaloid by fungal cells. The reversible excretion of aurantioclavine in P. nalgiovense indicates that this process is regulatory and depends on the growth parameters and the physiological state of the fungus.     

The Penicillium commune Thom and Penicillium clavigerum Demelius fungi--fumigaclavines A and B producers

The type strains Penicillium clavigerum VKM F-447 and P. commune VKM F-3233 are found to produce fumigaclavines A and B. Of the seven other strains of these species, only two strains, P. commune VKM F-3088 and F-3491, possess the ability to synthesize these alkaloids. It is suggested that the five other strains under study either lost such an ability or require very specific conditions for the synthesis of these alkaloids.     

Selection of ergot alkaloid producers by induced mutagenesis

Using the induced mutagenesis technique, A series of genetically modified Claviceps sp. VKM F-2609 strains that display high levels of agroclavine and elymoclavine synthesis were selected by induced mutagenesis. Compared to the parent strain, c106 displayed a 40-fold higher level of agroclavine synthesis, and c66 displayed an eightfold higher level of elymoclavine synthesis. The levels of synthesis of other alkaloids were decreased in these strains. The effects of various carbohydrates on the strain growth and ergot alkaloid biosynthesis was then investigated in both the parent strain and c106. The largest amount of agroclavine was synthesized by c106 strain growing on a medium with maltose.     

Production of extracellular H2O2 and L-lysine-α-oxidase during bulk growth of the fungus Trichoderma cf. aureoviride Rifai VKM F-4268D under salt stress

In this article, we report on Some physiological aspects of the synthesis of extracellular L-lysine-??-oxidase (LO) by the fungus Trichoderma cf. aureoviride Rifai VKM F-4268D under salt stress conditions and discuss the possible role of this enzyme for the producer. It has been shown that The synthesis of extracellular LO and proteolytic enzymes is induced in the fungus T. cf. aureoviride Rifai VKM F-4268D during submerged cultivation on wheat bran under salt stress. It has been shown that LO biosynthesis is accompanied by H₂O₂ accumulation in the growth medium. It seems that the extracellular LO synthesis followed by hydrogen peroxide production under stress conditions provides an adaptive advantage for the producer fungus in its competition with other organisms.     

Conversion of 1-benzoylindole by Aspergillus strains

Biotransformation of 1-benzoylindole (BI) by the strains Aspergillus flavus VKM F-1024 and Aspergillus oryzae VKM F-44 was studied. The major metabolites isolated were identified as 4-hydroxyindole (4-HI), 5-hydroxyindole (5-HI), 4-hydroxy-1-benzoylindole, 4-hydroxy-1-(4′-hydroxy)-benzoylindole and indole. The structure of the metabolites was determined by mass spectrometry and proton nuclear magnetic resonance spectroscopy. The pathways of BI metabolism via initial monohydroxylation at C-4 and C-5 followed by cleavage of the benzoyl substituent to yield 4-HI and 5-HI were proposed. Indole was formed as a by-product, and its role as a potent inhibitor of BI hydroxylation at C-4 and C-5 is discussed. Received: 22 June 1999 / Received revision: 6 December 1999 / Accepted: 12 December 1999