Physiological and biochemical characteristics of the genus <Emphasis Type="Italic">Penicillium</Emphasis> fungi as producers of ergot alkaloids and quinocitrinins

Four cultures of fungi of the genus Penicillium belonging to Furcatum Pitt subgenus, such as P. citrinum Thom, 1910; P. corylophilum Dierckx, 1901; P. fellutanum Biourge, 1923; and P. waksmanii Zaleski, 1927, produced the ergot alkaloids, namely, agroclavine-I, and epoxyagroclavine-I; their N-N-dimers, such as dimer of epoxyagroclavine-I and the mixed dimer of epoxyagroclavine-I and agroclavine-I; and also quinoline metabolites, namely, quinocitrinin A and quinocitrinin B. Physiological and biochemical characteristics of the producers were studied. Optimal conditions for the biosynthesis of metabolome components were determined. Zinc additive to the medium stimulated the biosynthesis of the ergot alkaloids in all cases; quinocitrinines production was increased only in P. citrinum, and that was suppressed in P. corylophinum, P. fellutanum, and P. waksmanii. This testifies that genes of the biosynthesis pathways are located in the different clusters of the producers.     

Transport of ergot alkaloids and quinocitrinins in the producing fungus Penicillium citrinum

The decrease in the concentration of alkaloids in the culture liquid of Penicillium citrinum grown to the early stationary phase was found to be due to the uptake of quinocitrinins and ergot alkaloids by fungal cells. The ability of the fungal mycelium to absorb autogenous quinocitrinins does not depend on the mycelium age, whereas its ability to absorb ergot alkaloids is higher in the young than in the 12-day-old mycelium. The uptake of exogenously added ergot alkaloids by the fungal mycelium is accompanied by excretion of intracellular quinocitrinins. The addition of quinocitrinins to the medium was found to exert different effects in different growth stages. Namely, the uptake of exogenously added quinocitrinins by the actively growing young mycelium inhibits the excretion of ergot alkaloids, but the excretion of ergot alkaloids by the 12- day-old mycelium occurs throughout the cultivation period. The excretion of both ergot alkaloids and quinocitrinins does not require energy.     

The effect of some amino acids on the growth and biosynthesis of ergot alkaloids and quinocitrinins in the fungus Penicillium citrinum Thom 1910

The effect of some amino acids, added to the medium either during inoculation or in the stationary growth phase, on the growth and biosynthesis of ergot alkaloids and quinocitrinins in the fungus Penicillium citrinum VKM FW-800 has been studied. Exogenously added amino acids were mostly utilized in primary metabolism. When added during inoculation, tryptophan and leucine virtually did not influence fungal growth and synthesis of the alkaloids, whereas the addition of isoleucine enhanced the biomass accumulation. When added in the stationary growth phase, tryptophan stimulated the synthesis of both ergot alkaloids and quinocitrinins. Leucine added in the stationary growth phase did not influence the synthesis of ergot alkaloids but inhibited the synthesis of quinocitrinins. Isoleucine inhibited the synthesis of both ergot alkaloids and quinocitrinins irrespective of the time of its addition to the medium.     

The effect of some amino acids on the growth and biosynthesis of ergot alkaloids and quinocitrinins in the fungus Penicillium citrinum Thom 1910

The effect of some amino acids, added to the medium either during inoculation or in the stationary growth phase, on the growth and biosynthesis of ergot alkaloids and quinocitrinins in the fungus Penicillium citrinum VKM FW-800 has been studied. Exogenously added amino acids were mostly utilized in primary metabolism. When added during inoculation, tryptophan and leucine virtually did not influence fungal growth and synthesis of the alkaloids, whereas the addition of isoleucine enhanced the biomass accumulation. When added in the stationary growth phase, tryptophan stimulated the synthesis of both ergot alkaloids and quinocitrinins. Leucine added in the stationary growth phase did not influence the synthesis of ergot alkaloids but inhibited the synthesis of quinocitrinins. Isoleucine inhibited the synthesis of both ergot alkaloids and quinocitrinins irrespective of the time of its addition to the medium.     

Use of polymerase chain reaction for searching for producers of ergot alkaloids from among microscopic fungi]

The potential of the polymerase chain reaction for the detection of ergot alkaloid producers among microscopic fungi of the genera Penicillium and Claviceps was evaluated. Twenty-three strains of various species of fungi with a previously studied capacity for alkaloid production were used. The internal fragment of the gene encoding 4-dimethylallyltryptophan synthase, the enzyme catalyzing the first step in the biosynthesis of ergot alkaloids, was amplified using degenerated primers. This approach revealed an about 1.2-kb specific DNA fragment in micromycetes synthesizing ergot alkaloids with complete tetracyclic ergoline system. Microorganisms that produce alkaloids with modified C or D ergoline rings, as well as alpha-cyclopiazonic acid, did not yield the PCR fragment of the expected size. This fragment was also not found in fungi incapable of ergot alkaloid production.     

Transport of ergot alkaloids and quinocitrinins in the producing fungus <Emphasis Type="Italic">Penicillium citrinum</Emphasis>

The decrease in the concentration of alkaloids in the culture liquid of Penicillium citrinum grown to the early stationary phase was found to be due to the uptake of quinocitrinins and ergot alkaloids by fungal cells. The ability of the fungal mycelium to take up autogenous quinocitrinins does not depend on the mycelium age, whereas its ability to take up ergot alkaloids is higher in the young than in the 12-day-old mycelium. The uptake of exogenously added ergot alkaloids by the fungal mycelium is accompanied by excretion of intracellular quinocitrinins. The addition of quinocitrinins to the medium was found to exert different effects in different growth stages. Namely, the uptake of exogenously added quinocitrinins by the actively growing young mycelium inhibits the excretion of ergot alkaloids, but the excretion of ergot alkaloids by the 12-day-old mycelium occurs throughout the cultivation period. The excretion of both ergot alkaloids and quinocitrinins does not require energy.     

Fungi of the genus Penicillium as producers of physiologically active compounds (Review)

Fungi of the genus Penicillium isolated from little studied habitats are able to synthesize both previously known and new physiologically active compounds with diverse structures. They include secondary metabolites of alkaloid nature, i.e., ergot alkaloids, diketopiperazines, quinolines, quinazolines, benzodiazepines, and polyketides. We discuss the use of profiles of secondary metabolites for taxonomy purposes. Studying the physicochemical characteristics of producers of biologically active compounds showed that the biosynthesis of alkaloids is initiated on the first days of cultivation and proceeds simultaneously with growth. The cyclic character of alkaloid accumulation was recorded related to the processes of alkaloid biosynthesis, excretion from cells, degradation in culture fluid, and consumption by cells. Synchronic variations in the concentrations of intracellular tryptophan and alkaloids are necessary for the regulation of the optimal quantity of tryptophan necessary for the culture.     

Fungus Penicillium citrinum, isolated from permafrost sediments, as a producer of ergot alkaloids and new quinoline alkaloids quinocitrinines

Quinocitrinines and ergot alkaloids are synthesized by the strain Penicillium citrinum VKM FW-800 as the culture grows. The major part of these secondary metabolites are secreted into the medium. In the phase of growth deceleration, these metabolites were partly absorbed by the producer cells. Zinc ions stimulated both the primary and secondary metabolic processes. Addition of this microelement into the culture medium stimulated biomass accumulation and the synthesis of clavine alkaloids and quinocitrinines.     

Effect of the exogenous tryptophan on biosynthesis of ergot alkaloids in Penicillium sizovae

Various concentrations of exogenous L and D-tryptophan as well as of their analogue D,L-6-methyltryptophan were added to the growth medium of Penicillium sizovae during its inoculation and after the active growth of the fungus was over. The authors studied the effect of these compounds on the accumulation of exocellular alkaloids and biomass as well as on the synthesis of proteins, the content of free tryptophan in the cells, and the activity of tryptophan synthetase. As was shown in experiments using labeled tryptophan, this amino acid is a direct precursor of alkaloids in the culture.     

Physiological status of submergedClaviceps during enzymic assembly of ergot alkaloids

Ergot alkaloids are formed only for arelatively brief time during the lifespan of the culture and under conditions of reduced proliferation. They cannot be taken for waste products of general metabolism. Ergot strains are capable of carrying out all the simple steps of the anthranilic acid—tryptophan cycles. Alkaloids influence activities of certain enzymes of primary metabolism in the ergot mycelium,e.g. tryptophan synthetase, acetyl-CoA carboxylase, citrate synthase, isocitrate lyase, and malate synthase. Ergot alkaloids do not belong to a group of physiologically inert secondary metabolites. A tentative scheme of the enzymic assembly of the ergoline nucleus is presented. The increased yield of ergoline alkaloids may be attributed to the following points: (1) Unbalnced growth of the culture. (2) Support of competition of fatty acids and alkaloid biosynthesis for acetyl-CoA. (3) Decreased activities of tricarboxylic acid and glyoxylate cycles. (4) Positive association between the rate of protein turnover and alkaloid formation. (5) Stimulation of both tryptophan synthesis and degradation via kynurenine—anthranilate. (6) Regulation of tryptophan-histidine cross-pathway. (7) Continuous control of the alkaloid level during fermentation.     

Rugulosuvines A and B--diketopiperazine alkaloids from Penicillium rugulosum and Penicillium piscarium fungi]

Two diketopiperazine alkaloids, rugulosuvines A and B (tryptophan and phenylalanine are precursors), were isolated and purified from the culture liquid of Penicillium rugulosum VKM F-352 and Penicillium piscarium VKM F-325 fungi. Physical and physicochemical studies showed the absolute structure of rugulosuvine A. The absolute structure of rugulosuvine B was demonstrated to be similar to that of rugulosuvine A.     

New producers of biologically active compounds--fungal strains of the genus Penicillium isolated from permafrost

Screening of producers of secondary metabolites was carried out among 25 fungal strains of Penicillium genus isolated from permafrost in Arctic and Antarctic regions and Kamchatka. Nearly 50% of the investigated strains synthesize biologically active substances of alkaloid nature: ergot alkaloids, diketopiperazines, and quinoline derivatives. A large group of the identified metabolites belongs to mycotoxins. A strain of Penicillium waksmanii was found producing epoxiagroclavine-I and quinocitrinins. The main physiological and biochemical characteristics of this producer were investigated.     

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).     

Biotechnology of ergot alkaloids

The ergot alkaloids are a group of pharmacologically active compounds produced primarily by species of Claviceps, a fungal parasite of grasses and cereals.

Ergot is of historical interest: medieval midwives collected the fungus from naturally infected plants and used it in the induction of childbirth and in the control of postpartum bleeding. Many additional uses of the ergot alkaloids have been discovered since then, and yet the method of production has persisted to this day basically unchanged.

However, submerged culture of Claviceps is starting to supersede the use of systematically infected rye and a number of new opportunities, including the development of high yielding fungal strains and the production of semi-synthetic alkaloids, have become apparent.     

Biotechnology and genetics of ergot alkaloids

Ergot alkaloids, i.e. ergoline-derived toxic metabolites, are produced by a wide range of fungi, predominantly by members of the grass-parasitizing family of the Clavicipitaceae. Naturally occurring alkaloids like the D-lysergic acid amides, produced by the "ergot fungus" Claviceps purpurea, have been used as medicinal agents for a long time. The pharmacological effects of the various ergot alkaloids and their derivatives are due to the structural similarity of the tetracyclic ring system to neurotransmitters such as noradrenaline, dopamine or serotonin. In addition to "classical" indications, e.g. migraine or blood pressure regulation, there is a wide spectrum of potential new applications of this interesting group of compounds. The biotechnology of ergot alkaloids has a long tradition, and efficient parasitic and submerse production processes have been developed; the biochemistry of the pathway and the physiology of production have been worked out in detail. The recent identification of a cluster of genes involved in ergot alkaloid biosynthesis in C. purpurea and the availability of molecular genetic techniques allow the development of strategies for rational drug design of ergoline-related drugs by enzyme engineering and by biocombinatorial approaches.     

Physiological and biochemical contributions to the taxonomy of the genus prototheca

Five physiological and biochemical characters, which had proved to be valuable for the taxonomy of the genus Chlorella, were studied in the genus Prototheca. There is no hydrogenase activity and no liquefaction of gelatin. Most strains are very acidtolerant (limit of growth at pH 2.0 or 2.5) and very salt-tolerant (limit of growth at 4 or 5% NaCl). Two strains grow well at 38°C. The 16 strains, which were previously assigned to seven taxa, fall into four different groups. Our results tend to support the assumption that Prototheca might be related to Chlorella protothecoides.