Screening for Ergot Alkaloid Producers among Microscopic Fungi by Means of the Polymerase Chain Reaction

The potential of the polymerase chain reaction for the detection of ergot alkaloid producers among microscopic fungi of the generaPenicilliumand Clavicepswas 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 degenerate 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 -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.     

Secondary metabolite biosynthesis in cultured cells of Catharanthus roseus (L.) G. Don immobilized by adhesion to glass fibres

Summary Suspension-cultured cells of Catharanthus roseus (L.) G. Don were immobilized on glass fibre mats and cultivated in shake flasks. The highly-aggregated immobilized cells exhibited a slower growth rate and accumulated reduced levels of tryptamine and indole alkaloids, represented by catharanthine and ajmalicine, in comparison to cells in suspension. The increased total protein synthesis in immobilized cells suggests a diversion of the primary metabolic flux toward protein biosynthetic pathways and away from other growth processes. In vitro assays for the specific activity of tryptophan decarboxylase (TDC) and tryptophan synthase (TS) suggest that the decreased accumulation of tryptamine in immobilized cells was due to reduced tryptophan biosynthesis. The specific activity of TDC was similar in immobilized and suspension-cultured cells. However, the expression of TS activity in immobilized cells was reduced to less than 25% of the maximum level in suspension-cultured cells. The reduced availability of a free tryptophan pool in immobilized cells is consistent with the reduced TS activity. Reduced tryptamine accumulation, however, was not responsible for the decreased accumulation of indole alkaloids in immobilized cells. Indole alkaloid accumulation increased to a similar level in immobilized and suspension-cultured cells only after the addition of exogenous secolaganin to the culture medium. The addition of tryptophan resulted in increased accumulation of tryptamine, but had no effect on indole alkaloid levels. Reduced biosynthesis of secologanin, the monoterpenoid precursor to indole alkaloids, in immobilized cells is suggested. Immobilization does not appear to alter the activity of indole alkaloid biosynthetic enzymes in our system beyond, and including, strictosidine synthase. Offprint requests to: P. J. Facchini     

Stability of alkaloid production in cell suspension cultures of Tabernaemontana divaricata during long-term subculture

Two cell lines of Tabernaemontana divaricata cell suspension culture with different growth and alkaloid production profiles were transferred to the same medium. During 30 subcultures the changes in growth and alkaloid production were followed and compared to those of the original cell lines. The presence of NAA and BAP in the medium resulted in an increase of biomass and alkaloid yield. The effect on the growth proved to be stable during these 30 subcultures. Alkaloid production showed a maximum in the 4th subculture after the change of the medium, and stabilized on a higher level than found in the original cell lines. During some growth cycles also the activities of tryptophan decarboxylase (TDC), strictosidine synthase (SSS), and phenylalanineammonia-lyase (PAL) were measured. In both the original cell lines and the derived cell lines, growth and alkaloid production proved to be stable all through the experiment, although the derived cell lines had a period of adaptation to the new medium with increased productivity.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - BAP benzylaminopurine - DW dry weight - TDC tryptophan decarboxylase - SSS strictosidine synthase - PAL phenylalanineammonia-lyase - PAT phenylalanineammonia-transaminase     

Investigations into the occurrence of alkaloids in ergot and single sclerotia from the 2007 and 2008 harvests

As a contribution to the occurrence of ergot alkaloids in ergot from German rye and triticale, samples from the 2007 and 2008 harvests were analyzed. Twelve alkaloids—six pairs of main alkaloids and their corresponding epimers—were determined in extracts prepared under alkaline conditions by HPLC with fluorescence detection without preceding purification. The total alkaloid content was found to be 0.03–0.18% in ergot from rye (n = 19) and 0.06–0.22% in ergot from triticale (n = 4), respectively. Furthermore, single sclerotia (n = 40) were investigated in terms of alkaloid content and distributional pattern. The main alkaloids in ergot were ergocristine, ergotamine and ergocornine, although the alkaloid composition was highly variable. Presented in part at the 30th Mykotoxin-Workshop, Utrecht, The Netherlands, April 28–30, 2008     

Molecular characterization of a seed transmitted clavicipitaceous fungus occurring on dicotyledoneous plants (Convolvulaceae)

Ergoline alkaloids (syn. ergot alkaloids) are constituents of clavicipitaceous fungi (Ascomycota) and of one particular dicotyledonous plant family, the Convolvulaceae. While the biology of fungal ergoline alkaloids is rather well understood, the evolutionary and biosynthetic origin of ergoline alkaloids within the family Convolvulaceae is unknown. To investigate the possible origin of ergoline alkaloids from a plant-associated fungus, 12 endophytic fungi and one epibiotic fungus were isolated from an ergoline alkaloid-containing Convolvulaceae plant, Ipomoea asarifolia Roem. & Schult. Phylogenetic trees constructed from 18S rDNA genes as well as internal transcribed spacer (ITS) revealed that the epibiotic fungus belongs to the family Clavicipitaceae (Ascomycota) whereas none of the endophytic fungi does. In vitro and in vivo cultivation on intact plants gave no evidence that the endophytic fungi are responsible for the accumulation of ergoline alkaloids in I. asarifolia whereas the epibiotic clavicipitaceous fungus very likely is equipped with the genetic material to synthesize these compounds. This fungus resisted in vitro and in vivo cultivation and is seed transmitted. Several observations strongly indicate that this plant-associated fungus and its hitherto unidentified relatives occurring on different Convolvulaceae plants are responsible for the isolated occurrence of ergoline alkaloids in Convolvulaceae. This is the first report of an ergot alkaloid producing clavicipitaceous fungus associated with a dicotyledonous plant.Data deposition: The sequences reported in this paper have been deposited in the GenBank (accession numbers are given in the text and in Fig. 3) Dedicated to Dr. Dr. h. c. mult. Albert Hofmann, the great pioneer of ergot research, on the occasion of his 100th birthday     

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.     

Positive feedback effect of benzodiazepine alkaloids on enzymes of the aromatic pathway

Abstract Penicillium cyclopium produces benzodiazepine alkaloids from l -phenylalanine and anthranilate. The biosynthesis of both precursors involves the enzymes of the shikimate pathway DAHP synthase, chorismate mutase and anthranilate synthase, the latter two competing for the common substrate chorismate. After the cultures reached the phase of alkaloid production, the in vitro measurable activities of these three enzymes could be increased by adding the alkaloids during incubation. The stimulation is most pronounced with anthranilate synthase, whose activity most probably limits the rate of alkaloid formation. It is not seen with tryptophan synthase which is not involved in the formation of alkaloid precursors. The data suggest a far reaching feedback activation, coordinating precursor biosynthesis with the formation of secondary product.     

Proteome analysis of the medicinal plant <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

A proteomic approach is undertaken aiming at the identification of novel proteins involved in the alkaloid biosynthesis of Catharanthus roseus. The C. roseus cell suspension culture A11 accumulates the terpenoid indole alkaloids strictosidine, ajmalicine and vindolinine. Cells were grown for 21 days, and alkaloid accumulation was monitored during this period. After a rapid increase between day 3 and day 6, the alkaloid content reached a maximum on day 16. Systematic analysis of the proteome was performed by two-dimensional polyacrylamide gel electrophoresis. After day 3, the proteome started to change with an increasing number of protein spots. On day 13, the proteome changed back to roughly the same as at the start of the growth cycle. 88 protein spots were selected for identification by mass spectrometry (MALDI-MS/MS). Of these, 58 were identified, including two isoforms of strictosidine synthase (EC 4.3.3.2), which catalyzes the formation of strictosidine in the alkaloid biosynthesis; tryptophan synthase (EC 4.1.1.28), which is needed for the supply of the alkaloid precursor tryptamine; 12-oxophytodienoate reductase, which is indirectly involved in the alkaloid biosynthesis as it catalyzes the last step in the biosynthesis of the regulator jasmonic acid. Unique sequences were found, which may also relate to unidentified biosynthetic proteins.     

Cell-pool tryptophan phases in ergot alkaloid fermentation

Three cell-pool tryptophan phases are recorded as characteristics of the alkaloid fermentation byClaviceps paspali grown on a simple defined medium without tryptophan. Within the early phase designated “tryptophan down” the alkaloid-biosynthetic activity of the mycelium attains the maximum, protein synthesis is reduced and extracellular proteases are formed. Cell-pool tryptophan level (b) drops, tryptophan synthetase activity (c) intensifies and sums of logb+logc after different time intervals remain constant. In the subsequent “tryptophan up” phase tryptophan level (b) increases, alkaloid yield (a) becomes a function of time and reaches the top level still tolerable by tryptophan synthetase. The difference of the logb—logc is constant. The tryptophan synthetase diminishes its activity simultaneously with the alkaloid-biosynthetic activity of the mycelium. The district between the “tryptophan down” and “tryptophan up” phase is an especially promising target for the investigation of the regulation of alkaloid formation and continuous fermentation of these compounds. During the third, i.e. “tryptophan over” phase, cell-pool tryptophan accumulates and attains a concentration exerting a negative effect on the alkaloid biosynthesis.     

Quantitative changes of the alkaloid complex in a submerged culture ofClaviceps paspali

Claviceps paspali FA produced high concentrations of alkaloid under submerged conditions. Their production was found to depend on the developmental stage and treatment of the filamentous culture inoculum. A medium containing Bacto-peptone with a constant composition of amino acids was selected for the preparation of the inoculum. A two-week fermentation in a synthetic medium with mannitol at 24 ± 1 °C resulted in an increased production of total alkaloids from the original value of 100–200 μg/mL to more than 2 000 μg/mL. Addition of tryptophan did not further increase the production of alkaloids but resulted in changes of the spectrum of some metabolites. 2,3-Dihydroxybenzoic acid accompanied the alkaloids in the fermentation medium. α-Hydroxyethyllysergamide was the predominant component of extracellular alkaloids (80 % in the first days of fermentation). During fermentation the level of this alkaloid continuously decreased while the concentration of the accompanying alkaloids,i.e. lysergamide and the corresponding minor isomers, increased. An erratum to this article is available at .     

The ergot alkaloid gene cluster: Functional analyses and evolutionary aspects

Ergot alkaloids and their derivatives have been traditionally used as therapeutic agents in migraine, blood pressure regulation and help in childbirth and abortion. Their production in submerse culture is a long established biotechnological process. Ergot alkaloids are produced mainly by members of the genus Claviceps, with Claviceps purpurea as best investigated species concerning the biochemistry of ergot alkaloid synthesis (EAS). Genes encoding enzymes involved in EAS have been shown to be clustered; functional analyses of EAS cluster genes have allowed to assign specific functions to several gene products. Various Claviceps species differ with respect to their host specificity and their alkaloid content; comparison of the ergot alkaloid clusters in these species (and of clavine alkaloid clusters in other genera) yields interesting insights into the evolution of cluster structure. This review focuses on recently published and also yet unpublished data on the structure and evolution of the EAS gene cluster and on the function and regulation of cluster genes. These analyses have also significant biotechnological implications: the characterization of non-ribosomal peptide synthetases (NRPS) involved in the synthesis of the peptide moiety of ergopeptines opened interesting perspectives for the synthesis of ergot alkaloids; on the other hand, defined mutants could be generated producing interesting intermediates or only single peptide alkaloids (instead of the alkaloid mixtures usually produced by industrial strains).     

Alkaloid formation by habituated and tumorous cell suspension cultures of Catharanthus roseus

Habituated and tumorous Catharanthus roseus cells grown in the absence of hormones accumulated indole alkaloids. Total alkaloids and alkaloid pattern were the same when cells were cultured in medium without hormones or in alkaloid production medium with and without indole acetic acid. Treatment of cells with Pythium homogenate as elicitor did not increase total alkaloids or change the pattern of alkaloids produced. When either habituated or tumorous cells were grown in 1B5 medium after Gamborg et al (1968) containing 2,4-dichlorophenoxyacetic acid (2,4-D), their capacity to accumulate alkaloids decreased with time. The levels of tryptophan decarboxylase (TDC) and strictosidine synthase (SS) specific activities were constant throughout growth except when cells were exposed to 2,4-D in 1B5 medium, where enzyme activities declined in step with the decrease in alkaloid accumulation. Neither habituated nor tumorous cell suspension cultures accumulated vindoline, nor could they be induced to produce this alkaloid by any of the given treatments.NRCC No. 27514     

Sugar and phosphate metabolism and alkaloid production phases in submerged cultures of two Claviceps strains

Summary In submerged cultures of Claviceps sp. CP II, elymoclavine was synthesized only by the growing mycelium (phase P1), whereas cultures of C. purpurea strain 129 produced agroclavine after vegetative growth had also ceased (phase P2). In strain CP II, the peak of activity of malate dehydrogenase, glucose-6-phosphate dehydrogenase and phosphatases was related to the time of maximum growth rate and alkaloid production. Citrate synthase activity paralleled the course of alkaloid synthesis. Strain 129 exhibited a further activity peak of the same magnitude during phase P2. ATP levels in both cultures corresponded to the pattern of change in enzyme activities. Strain CP II contained roughly twice as much orthophosphate and ATP in its cells as strain 129 and exhibited higher average activity of glucose-6-phosphate dehydrogenase. It follows from these results that alkaloid synthesis requires the processes of primary metabolism, even when it occurs after active growth of the culture has ceased. Cultures producing alkaloids oxidized at C-8 exhibit higher glucose-6-phosphate dehydrogenase activity, probably because of a higher NADPH consumption.     

Tryptophan decarboxylase strictosidine synthase and alkaloid production by Cinchona ledgeriana suspension cultures

The activities of tryptophan decarboxylase (TDC) and strictosidine synthase (SS) have been compared with the production of quinoline alkaloids in light-grown and dark-grown suspension cultures of Cinchona ledgeriana transformed with Agrobacterium tumefaciens. Enzyme activities and alkaloid production were both substantially greater in the dark-grown cultures than in the light-grown cultures. Under the conditions of assay, SS was in all cases at least 80 times more active than TDC and did not vary very substantially over a 22-day culture period. In contrast, TDC activity in the dark-grown cells rose rapidly to a maximum after 13 days and then declined. Total alkaloid production was largely growth-related, but the cellular alkaloid content declined sharply (on account of release into the medium) within the first 4 days after subculture, prior to the rise in TDC activity. TDC activity over the 22-day period was only 2-to 3-fold greater than that required to account for alkaloid production and is therefore potentially rate-limiting under in vivo conditions.     

Biosynthesis and accumulation of ergoline alkaloids in a mutualistic association between Ipomoea asarifolia (Convolvulaceae) and a clavicipitalean fungus

Ergoline alkaloids occur in taxonomically unrelated taxa, such as fungi, belonging to the phylum Ascomycetes and higher plants of the family Convolvulaceae. The disjointed occurrence can be explained by the observation that plant-associated epibiotic clavicipitalean fungi capable of synthesizing ergoline alkaloids colonize the adaxial leaf surface of certain Convolvulaceae plant species. The fungi are seed transmitted. Their capacity to synthesize ergoline alkaloids depends on the presence of an intact differentiated host plant (e.g. Ipomoea asarifolia or Turbina corymbosa [Convolvulaceae]). Here, we present independent proof that these fungi are equipped with genetic material responsible for ergoline alkaloid biosynthesis. The gene (dmaW) for the determinant step in ergoline alkaloid biosynthesis was shown to be part of a cluster involved in ergoline alkaloid formation. The dmaW gene was overexpressed in Saccharomyces cerevisiae, the encoded DmaW protein purified to homogeneity, and characterized. Neither the gene nor the biosynthetic capacity, however, was detectable in the intact I. asarifolia or the taxonomically related T. corymbosa host plants. Both plants, however, contained the ergoline alkaloids almost exclusively, whereas alkaloids are not detectable in the associated epibiotic fungi. This indicates that a transport system may exist translocating the alkaloids from the epibiotic fungus into the plant. The association between the fungus and the plant very likely is a symbiotum in which ergoline alkaloids play an essential role.     

The Effects of Calcium and the Ionophore A23I87 on Modulation, Nitrogen Fixation and Growth of Soybeans

Tryptophan synthase in Phycomyces blakesleeanus. Part II: Activity of tryptophan synthase in Phycomyces blakesleeanus depending on the light and the content of zinc ions in the culture medium Five-day-old cultures of Phycomyces blakesleeanus show notice-able differences in the phenotype, depending on the culture conditions (permanent light, permanent dark, zinc deficiency, zinc sufficiency) and related to the distribution of tryptophan synthase activity between mycelium and sporangiophores. Permanent light and the presence of zinc ions in the medium during culturing have an antagonistic influence on the tryptophan synthase. The activity of the enzyme is being reduced in the sporangiophores and increased in the mycelium by the influence of light, while zinc ions in the culture medium increase the activity in the sporangiophores at simultaneous reduction in the mycelium. The importance of tryptophan synthase and tryptophan for the development of the fungus in relation to the metabolism of indole acetic acid is discussed.     

Biotechnological potential of the ethylmalonyl-CoA pathway

The ethylmalonyl-CoA pathway is central to the carbon metabolism of many α-proteobacteria, like Rhodobacter sphaeroides and Methylobacterium extorquens as well as actinomycetes, like Streptomyces spp. Its function is to convert acetyl-CoA, a central carbon intermediate, to other precursor metabolites for cell carbon biosynthesis. In contrast to the glyoxylate cycle—another widely distributed acetyl-CoA assimilation strategy—the ethylmalonyl-CoA pathway contains many unique CoA-ester intermediates, such as (2R)- and (2S)-ethylmalonyl-CoA, (2S)-methylsuccinyl-CoA, mesaconyl-(C1)-CoA, and (2R, 3S)-methylmalyl-CoA. With this come novel catalysts that interconvert these compounds. Among these unique enzymes is a novel carboxylase that reductively carboxylates crotonyl-CoA, crotonyl-CoA carboxylase/reductase, and (3S)-malyl-CoA thioesterase. The latter represents the first example of a non-Claisen condensation enzyme of the malate synthase superfamily and defines a new class of thioesterases apart from the hotdog-fold and α/β-fold thioesterases. The biotechnological implications of the ethylmalonyl-CoA pathway are tremendous as one looks to tap into the potential of using these new intermediates and catalysts to produce value-added products.     

Effect of differentiation on the regulation of indole alkaloid production in Catharanthus roseus hairy roots

In vitro cultures of hairy root derived from Catharanthus roseus accumulate higher levels of indole alkaloids than cell suspension cultures. Hairy roots were interconverted to undifferentiated cells by manipulation of the culture medium. When the concentration of micronutrients in the culture medium was five times that of Phillips and Collins (1979) medium, cell suspensions formed from the hairy roots. The alkaloid content was five times lower in the cell suspensions than in the control, but upon regeneration of the roots the alkaloid content regained its original level. The formation of cell suspensions from hairy roots was also accompanied by a reduction in tryptophan decarboxylase and the strictosidine synthase activity to less than 5% and 30%, respectively. 3-Hydroxymethylglutaryl coenzyme A reductase activity was the same in the cell suspension and in the regenerated line. Received: 12 February 1998 / Revision received: 21 May 1998 / Accepted: 5 June 1998     

Induction of ajmalicine formation and related enzyme activities in Catharanthus roseus cells: effect of inoculum density

In Catharanthus roseus cell cultures the time courses of four enzyme activities, tryptophan decarboxylase (TDC), strictosidine synthase (SSS), geraniol-10-hydroxylase (G10H) and anthranilate synthase (AS), and alkaloid accumulation were compared under two different culture conditions (low-inoculum density and high-inoculum density on induction medium) and a control on growth medium. In growth medium a transient increase in TDC activity was first observed after which G10H reached its maximum activity; only tryptamine accumulated, no ajmalicine could be detected. Apparently, a concerted induction of enzyme activities is required for ajmalicine formation. Cells inoculated in induction medium showed such a concerted induction of AS, TDC and G10H activities. After 30 days the low-density culture had accumulated six times more ajmalicine (in moles/g) than the high-density culture. Thus, increase in biomass concentration (high-density cultures) did not enhance the total alkaloid production. The major differences observed in enzyme levels between high-and low-density cultures were in the AS and TDC activities, which were two to three times higher in the low-density culture, indicating that there is a positive correlation between ajmalicine formation and AS and TDC activities.Biotechnology Delft Leiden, Project Group Plant Cell Biotechnology Correspondence to: R. Verpoorte