Isolation and Characterization of Vacuoles from the Ergot Fungus Claviceps purpurea

Protoplasts of Claviceps purpurea were prepared by treatment of mycelium with a lytic mixture of snail gut enzyme and cellulase from Trichoderma viride. Such protoplasts could be efficiently lysed by Triton X-100 treatment at high osmotic pressure without Ca²⁺ or Mg²⁺, allowing the release of intact vacuoles in high yields. Vacuoles obtained from cells grown in modified Vogel medium (vegetative-type cells not producing alkaloids) were isolated and purified by centrifugation from a 5% Ficoll 400 (wt/vol) phase into the interphase between two layers, one containing 0.25 M each of mannitol and sucrose, and one containing 0.5 M mannitol. Vacuoles derived from cells grown in a medium favoring ergot alkaloid synthesis (sclerotia-like cells) were isolated by gentle centrifugation of filtered protoplast lysates without addition of Ficoll 400. Biochemical analyses of the vacuole fraction isolated from either kind of cell revealed their function as compartments harboring several hydrolytic enzymes. However, the enrichment of free amino acids in vacuoles of sclerotia-like cells was less pronounced than that in vacuoles of vegetative-type cells, indicating a difference in metabolic compartmentation in the two types of cells.     

Alkaloid Cluster Gene ccsA of the Ergot Fungus Claviceps purpurea Encodes Chanoclavine I Synthase,a Flavin Adenine Dinucleotide-Containing Oxidoreductase Mediating the Transformation of N-Methyl-Dimethylallyltryptophan to Chanoclavine I

Ergot alkaloids are indole-derived secondary metabolites synthesized by the phytopathogenic ascomycete Claviceps purpurea. In wild-type strains, they are exclusively produced in the sclerotium, a hibernation structure; for biotechnological applications, submerse production strains have been generated by mutagenesis. It was shown previously that the enzymes specific for alkaloid biosynthesis are encoded by a gene cluster of 68.5 kb. This ergot alkaloid cluster consists of 14 genes coregulated and expressed under alkaloid-producing conditions. Although the role of some of the cluster genes in alkaloid biosynthesis could be confirmed by a targeted knockout approach, further functional analyses are needed, especially concerning the early pathway-specific steps up to the production of clavine alkaloids. Therefore, the gene ccsA, originally named easE and preliminarily annotated as coding for a flavin adenine dinucleotide-containing oxidoreductase, was deleted in the C. purpurea strain P1, which is able to synthesize ergot alkaloids in axenic culture. Five independent knockout mutants were analyzed with regard to alkaloid-producing capability. Thin-layer chromatography (TLC), ultrapressure liquid chromatography (UPLC), and mass spectrometry (MS) analyses revealed accumulation of N-methyl-dimethylallyltryptophan (Me-DMAT) and traces of dimethylallyltryptophan (DMAT), the first pathway-specific intermediate. Since other alkaloid intermediates could not be detected, we conclude that deletion of ccsA led to a block in alkaloid biosynthesis beyond Me-DMAT formation. Complementation with a ccsA/gfp fusion construct restored alkaloid biosynthesis. These data indicate that ccsA encodes the chanoclavine I synthase or a component thereof catalyzing the conversion of N-methyl-dimethylallyltryptophan to chanoclavine I.The ergot fungus Claviceps purpurea is a phytopathogenic ascomycete which infects the ears of several grasses, replacing the ovary and producing a hibernation structure, the so-called sclerotium, in which the ergot alkaloids are formed. These substances show a high level of structural homology to some neurotransmitters like serotonin and dopamine and can therefore bind to the same receptors in the central nervous system (CNS), which is the basis for the application of ergot alkaloids in a variety of clinical conditions (15).The biochemistry of ergot alkaloid biosynthesis was first investigated by isolation of intermediates and postulation of a hypothetical pathway as well as enzymes needed for the successive biosynthetic steps of the production (Fig. ​(Fig.1).1). Most of the data were collected by pursuing the fate of radiolabeled precursors in feeding experiments (4). The first enzyme which could be assigned to alkaloid production was dimethylallyltryptophan synthetase (DMATS), which is the key enzyme of the pathway and is encoded by the gene dmaW (18). These analyses were performed with a Claviceps fusiformis strain, but a homolog of dmaW ({"type":"entrez-nucleotide","attrs":{"text":"AY259840","term_id":"32402653","term_text":"AY259840"}}AY259840) possessing a similar function could also be isolated in C. purpurea, as was confirmed by a knockout approach (N. Lorenz and P. Tudzynski, unpublished data). Using genome walking combined with cDNA screening, a 68.5-kb genomic region surrounding dmaW could be sequenced and revealed 14 open reading frames (ORFs) (putative genes) encoding, among others, nonribosomal peptide synthetases (NRPSs), a putative catalase, a CYP450-1 monooxygenase, a putative methyltransferase, and several oxidoreductases (6, 13, 19) (Fig. ​(Fig.2).2). Some of these genes were functionally and biochemically analyzed by a gene replacement approach which revealed their function within the pathway (2, 5, 7). However, there is still a deficit in functional analyses, especially with respect to the early steps within this pathway. The conversion from N-methyl-dimethylallyltryptophan (Me-DMAT) to agroclavine via chanoclavine I and chanoclavine I aldehyde includes successive oxidation and reduction steps mediated by a specific class of enzymes, the oxidoreductases (15) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Biosynthetic pathway of the ergot alkaloid biosynthesis of C. purpurea. Genes analyzed so far have been assigned to the corresponding enzyme at the corresponding position within the pathway. DMAPP, dimethylallyldiphosphate; DMAT, dimethylallyltryptophan; Me-DMAT, N-methyl-DMAT. (Adapted from reference 7 with permission of Wiley-VCH Verlag GmbH & Co. KGaA.)Open in a separate windowFIG. 2.Alkaloid biosynthesis gene cluster of C. purpurea. Highlighted in white is the gene of interest ccsA. (Adapted from reference 7 with permission of Wiley-VCH Verlag GmbH & Co. KGaA.)These enzymes are involved in the biosynthesis of many fungal secondary metabolites. A prominent example is the family of the cytochrome P450 monooxygenases (named after the characteristic peak of 450 nm when complexed with carbon monoxide). Cytochrome P450 (CYP450) monooxygenases catalyze the transfer of one oxygen atom from molecular oxygen to various substrates, mostly accomplished by the involvement of NAD(P)H as an electron donor. The eas cluster of C. purpurea also includes a gene encoding a CYP450 monooxygenase: cloA is involved in the oxidation of elymoclavine, leading to the formation of paspalic acid (7).No further monooxygenase-encoding genes seem to be present in the eas cluster, but several genes code for putative oxidoreductases (easA, easD, easE, easG, and easH). These oxidoreductases are most likely involved in the early steps within the pathway, but none of them has been functionally analyzed so far (15).We initiated a functional analysis of the putative oxidoreductase-encoding gene ccsA (formerly easE) (Fig. ​(Fig.2).2). The coding region of ccsA ({"type":"entrez-nucleotide","attrs":{"text":"AJ011965","term_id":"4499842","term_text":"AJ011965"}}AJ011965; 1,503 bp) is composed of two exons interrupted by an intron of 52 bp, yielding a coding capacity of 483 amino acids (aa). The gene product shows highest similarity to putative oxidoreductases of other ergot alkaloid-producing fungi: EasE of C. fusiformis (e⁻¹⁶⁰; {"type":"entrez-protein","attrs":{"text":"ABV57823","term_id":"157488556","term_text":"ABV57823"}}ABV57823), EasE of Neotyphodium lolii (e⁻¹¹⁸; {"type":"entrez-protein","attrs":{"text":"ABM91450","term_id":"124110194","term_text":"ABM91450"}}ABM91450) and CpoX1 of Aspergillus fumigatus (e⁻⁹⁶; {"type":"entrez-nucleotide","attrs":{"text":"XM_751049","term_id":"71002921","term_text":"XM_751049"}}XM_751049). Analyses of the protein sequence using the program PROSITE revealed a flavin adenine dinucleotide (FAD)-binding domain (pfam01565) spanning the region from amino acids 14 to 161 and a berberine bridge enzyme domain (BBE domain; pfam08031) from amino acids 412 to 457. The role of CcsA in the alkaloid biosynthesis pathway was investigated by knockout of the corresponding gene, followed by functional and biochemical analyses of the deletion mutants.     

An Old Yellow Enzyme Gene Controls the Branch Point between Aspergillus fumigatus and Claviceps purpurea Ergot Alkaloid Pathways

Ergot fungi in the genus Claviceps and several related fungal groups in the family Clavicipitaceae produce toxic ergot alkaloids. These fungi produce a variety of ergot alkaloids, including clavines as well as lysergic acid derivatives. Ergot alkaloids are also produced by the distantly related, opportunistic human pathogen Aspergillus fumigatus. However, this fungus produces festuclavine and fumigaclavines A, B, and C, which collectively differ from clavines of clavicipitaceous fungi in saturation of the last assembled of four rings in the ergoline ring structure. The two lineages are hypothesized to share early steps of the ergot alkaloid pathway before diverging at some point after the synthesis of the tricyclic intermediate chanoclavine-I. Disruption of easA, a gene predicted to encode a flavin-dependent oxidoreductase of the old yellow enzyme class, in A. fumigatus led to accumulation of chanoclavine-I and chanoclavine-I-aldehyde. Complementation of the A. fumigatus easA mutant with a wild-type allele from the same fungus restored the wild-type profile of ergot alkaloids. These data demonstrate that the product of A. fumigatus easA is required for incorporation of chanoclavine-I-aldehyde into more-complex ergot alkaloids, presumably by reducing the double bond conjugated to the aldehyde group, thus facilitating ring closure. Augmentation of the A. fumigatus easA mutant with a homologue of easA from Claviceps purpurea resulted in accumulation of ergot alkaloids typical of clavicipitaceous fungi (agroclavine, setoclavine, and its diastereoisomer isosetoclavine). These data indicate that functional differences in the easA-encoded old yellow enzymes of A. fumigatus and C. purpurea result in divergence of their respective ergot alkaloid pathways.Different classes of ergot alkaloids are produced by members of two distinct fungal lineages. Clavicipitaceous species, which include Claviceps spp. and Neotyphodium spp., are in the order Hypocreales and typically synthesize lysergic acid derivatives (13, 16, 18). These alkaloids have a double bond in the last assembled of four rings (D ring) of the tetracyclic ergoline ring structure. Ergot alkaloids are also produced by the distantly related opportunistic human pathogen Aspergillus fumigatus, a member of the order Eurotiales (8, 14, 16, 18). Ergot alkaloids of A. fumigatus are of the clavine class and differ from the more complex profile of Claviceps purpurea and Neotyphodium spp. One important distinction between the ergot alkaloids produced by these different fungi is the saturation of the fourth ring of the ergoline structure in A. fumigatus (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Structures and relationships of relevant ergot alkaloids. (A) Chanoclavine-I is oxidized to its aldehyde form before being incorporated into festuclavine (and downstream alkaloids) in A. fumigatus or agroclavine (and downstream alkaloids) in C. purpurea. (B) Conventional ring labeling and atom numbering referred to in the text.Several genes involved in the ergot alkaloid pathways of A. fumigatus and clavicipitaceous fungi are found clustered together in the genome of each species (3, 4, 6, 18, 23). These distantly related fungi are hypothesized to share several early pathway steps, after which the pathways diverge to yield distinct sets of ergot alkaloids (3, 13, 16). The gene dmaW, which encodes dimethylallyltryptophan (DMAT) synthase, catalyzes the prenylation of tryptophan that initiates the ergot alkaloid pathway in clavicipitaceous fungi (22, 25) and functions similarly in A. fumigatus (3, 24). The region surrounding this gene in A. fumigatus contains homologues of genes also found in Neotyphodium lolii and C. purpurea ergot alkaloid gene clusters (3, 4, 6). One of the shared genes, easA, is predicted to encode a member of the old yellow enzyme (OYE) family of oxidoreductases. Old yellow enzymes are flavin-containing oxidoreductases initially found in the brewer''s bottom yeast Saccharomyces carlsbergensis (26). Enzymes in this family use a reduced flavin cofactor and an active-site tyrosine residue to reduce the carbon-carbon double bond in an α/β-unsaturated aldehyde or ketone (7, 10). Subsequently, the enzymes require NADPH to restore the flavin cofactor to its reduced state. OYEs catalyze multiple reactions useful for both biotechnological and pharmaceutical applications; however, physiological roles and natural substrates for many of these enzymes presently are unknown (26). On the basis of the apparent need in the ergot alkaloid pathway of A. fumigatus for reduction of a carbon-carbon double bond in the intermediate chanoclavine-I-aldehyde, we hypothesized that the OYE-encoding gene easA is required for ergot alkaloid biosynthesis (3, 16). In this study, easA in A. fumigatus was disrupted and complemented to ascertain the role of its gene product in ergot alkaloid biosynthesis.     

Methods for Mutation and Selection of the Ergot Fungus

A new method is described in which the Salkowski reaction is used for the rapid selection of alkaloid-producing mutants of the ergot fungus. This method was used to investigate the influence of a second mutation with N-methyl-N′-nitro-N-nitrosoguanidine (NTG) on various mutants selected by a preliminary NTG mutation of Claviceps sp. strain SD 58. Three groups of mutants were used: high alkaloid producers, low alkaloid producers, and auxotrophs. Results indicated that a second mutation of all three types of mutants could improve alkaloid yield and vegetative vigor. In addition, a second mutation increased the frequency of auxotroph production. The difficulty of producing stable mutants from ergot strains that have multinucleated cells and that do not readily produce conidia in culture, such as an ergotamine-producing strain, was overcome by first forming protoplasts of the fungus and then subjecting them to the mutagen. Stable auxotrophs were obtained in this manner.     

Alkaloid production during the cultivation with shaking of Claviceps sp: Effects of asparagine

Among the nitrogen sources tested, asparagine stimulated alkaloid production maximally. Ammonium salts supported alkaloid production poorly. During the cultivation with shaking of Claviceps sp. strain SD-58 in asparagine containing medium, the activity of asparagine increased during the exponential growth (up to 8 days) with the intracellular accumulation of ammonium ions. Among the ammonia-assimilating enzymes we studied, NADP⁺-glutamate dehydrogenase (GDH) had a higher activity in the growth phase (up to 6 days), while in the intensive alkaloid producing phase (after 6 days) the activity of glutamine synthetase was higher. The latter was associated with increases in the intracellular level of tryptophan and alkaloid production.The levels of NADP⁺- and NAD⁺-alanine dehydrogenases and glutamate synthase were negligible.     

Isolation of the Ergot Strain Claviceps purpurea(Fr.) Tul. VKM-F-3662D Producing the Lactamic Alkaloid Ergocornam

A new ergot strain, VKM-F-3662D, producing lactamic alkaloid ergocornam with concomitant alkaloids valinamide and ergometrine, was isolated during selective work with sclerotium MS-462, which was obtained from ergocryptine ergot strain VKM-F-2642D. The structure of these alkaloids was determined by ¹H and ¹³C NMR.     

Relationship between the Claviceps Life Cycle and Productivity of Ergot Alkaloids

Abstract

The morphology, biochemistry, and physiology studies during development of Claviceps purpurea fungi clearly demonstrate that alkaloid synthesis is linked to a specific stage of the fungal life cycle. In nature, ergot alkaloids are synthesized in the course of developing sclerotia, while in submerged cultures, lacking sexual reproduction, alkaloid synthesis proceeds in sclerotia-like cells. Highly active submerged strains could be obtained by combination of mutagens with a different mode of action as well as by somatic hyphal anastomoses or efficient protoplast fusions to obtain the parasexual cycle. Fused strains not only retained the biosynthetic activity of parent strains but produced even much higher amounts of alkaloids. In our strains, the appropriate morphology always corresponded to high productivity. Furthermore, the form of cell differentiation was typical for each particular strain. When comparing active and inactive strains, measurements of qualitative and quantitative changes in mycelium composition revealed different metabolic patterns and certain characteristics necessary for efficient alkaloid production. Evaluation of activities of some enzymes from the central metabolic pathways, which generate the basic intermediates for ergot alkaloid synthesis also contributed to the overall knowledge of mechanisms involved.     

Optimization of the Medium and Cultivation Conditions of Penicillium roquefortii f39 Producing the Diketopiperazine Alkaloid Roquefortine

We optimized the medium for cultivation of Penicillium roquefortii f39, a producer of roquefortine. In this medium, the roquefortine yield increased 1.5–2-fold. An increase in roquefortine content was associated with high biomass yield, but not with an increase in biosynthetic activity of the mycelium. Direct correlation was found between extracellular roquefortine concentration and the amount of the inoculum. The use of sucrose in the inoculum medium allowed us to increase the concentration of roquefortine during fermentation to 90 mg/l.     

白腐菌混合菌降解木质素最佳条件的优化

通过正交试验对3种白腐菌混合菌降解竹材木质素的条件进行优化,结果表明,在温度为32℃、pH3.0、固体发酵时间20 d、培养液与竹材基质质量百分比110%时降解木质素的效率最高.在此基础上,研究了两种诱导剂对白腐菌混合菌降解木质素的影响.结果表明,两种诱导剂均能促进木质素的降解,其中H_2O_2在浓度1%时,木质素降解率高达62.9%,苯甲酸在浓度0.1%时,木质素降解率最高,为67.8%.     

Role of Glycols and Tweens in the Production of Ergot Alkaloids by Claviceps paspali

Several glycols and Tweens markedly stimulated the production of ergot alkaloids in submerged cultures of Claviceps paspali. The role of these compounds was investigated in shake flasks and bench-scale fermentors. 2,3-Butanediol was not utilized by the fungus, and 1,2-propanediol-1-(14)C was not incorporated into the alkaloids. Glycols and Tweens lowered the surface tension of the basal medium and promoted the utilization of metabolites. In the presence of glycols and Tweens, an increased uptake of labeled sorbitol and succinic acid took place, whereas the specific radioactivity of the alkaloids was not affected. These results indicated that glycols and Tweens are not involved directly in the biosynthetic process; they apparently acted as surface-active agents, facilitating transport of metabolites into the cells.     

The Fungus Penicillium citrinum Thom 1910 VKM FW-800 Isolated from Ancient Permafrost Sediments As a Producer of the Ergot Alkaloids Agroclavine-1 and Epoxyagroclavine-1

The study of the secondary metabolites of the relict strain Penicillium citrinum VKM FW-800 isolated from ancient Arctic permafrost sediments showed that this fungus produces agroclavine-1 and epoxyagroclavine-1, which are rare ergot alkaloids with the 5R,10S configuration of the tetracyclic ergoline ring system. The production of the alkaloids by the fungus showed a biphasic behavior, being intense in the phase of active growth and slowing down in the adaptive lag phase and in the stationary growth phase. The addition of zinc ions to the incubation medium led to a fivefold increase in the yield of the alkaloids. The alkaloid-producing Penicillium fungi isolated from different regions exhibited the same tendencies of growth and alkaloid production.     

Conditions for Cultivation of the Fungus Penicillium melinii UzLM-4 and Its Biosynthesis of Lipases

Cultivation of the fungus Penicillium melinii UzLM-4 on a Raistrick's medium of our own modification made it possible to increase the biosynthesis of lipases by three to four times. The following conditions ensured a high rate of synthesis of the extracellular lipase: age of the inoculum, 15 days; concentration of the inoculum, 15 × 10⁶ conidia per 100 ml medium; initial pH of the nutrient medium, 8.0; and cultivation in a shaker at 150 rpm and 25°C.     

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.     

Optimization of conditions for storage and cultivation of the fungus Claviceps sp.--a producer of the ergot alkaloid agroclavine

Conditions of agroclavine biosynthesis by the mutant Claviceps sp. strain s 106 were studied. The content of agroclavine was maximum (1.5-2 g/l) on days 15-16 of cultivation in the complex medium T25, containing sucrose, citric acid, and yeast extract. Agroclavine was the major component of the alkaloid fraction (90-95%). Storage of the culture at -70 degrees C in T25 supplemented by 7% glycerol provided a stable level of alkaloid formation.     

Optimization of the medium and conditions for cultivatingErwinia sp., a producer of fumarase and aspartase

Mathematical methods of experimental design were used to determine the optimal concentrations of nutrient medium components, aeration conditions, and pH providing for the maximum biomass yields, as well as fumarase and aspartase activities, during submerged cultivation ofErwinia sp. The data showed that different concentrations of carbon source (molasses) and pH of the nutrient medium were required to reach the maximum yields of fumarase and aspartase. Calculations suggested that the combination of these optimized factors would result in 3.2-, 3.4-, and 3.8-fold increases in theErwinia sp. biomass, aspartase activity, and fumarase activity yields, respectively. The experimental data were consistent with these estimates to 80% accuracy.     

海洋红树林内生真菌产抗肿瘤活性化合物1403C发酵条件的优化

旨在考察pH、溶解氧和剪切力对Halorosellinia sp.( No.1403)发酵过程的影响.结果表明,在发酵中后期及整个发酵过程中控制发酵液的pH恒定对菌体的生长影响不明显,但在不同发酵时期控制不同的恒定pH值对1403C的产量影响较大.仅在发酵的前24h控制恒pH7.0,有利于菌丝的生长,但抑制1403C的生物合成,菌体干重为对照组的159.1％,1403C的产量仅为对照组的29.4％;从48 h开始控制恒pH7.0,有利于1403C的生物合成,菌体干重量为对照组的94％,1403C的产量为对照组的123.2％;适量的溶解氧和剪切力有利于1403C的生物合成;从48 h开始提高剪切力可获得较高的1403C产量,是对照组的151.8％.综上所述,在发酵至48 h时开始控制恒pH7.0,并适当增大剪切力,在整个发酵过程中控制适当充足的溶解氧有利于1403C的合成.     

Optimization of Culture Medium for Sporulation and Biomass Production of a Nematophagous Fungus: Consideration of Nutritional and Environmental Conditions

This study reports the effects of various nutritional and environmental factors on sporulation and biomass of Paecilomyces lilacinus IPC‐P. These factors included carbon and nitrogen sources, carbon‐to‐nitrogen ratios, mineral elements and vitamins together with water potentials, temperatures, dark/light cycles and pH. On the basis of these results, together with a ‘two‐step’ cultivation and orthogonal method, the culture conditions for sporulation of this fungus were optimized. The spore suspension was inoculated on a basal medium (sucrose 19.00 g/l, soy peptone 4.06 g/l, K₂HPO₄ 1.00 g/l, KCl 0.50 g/l, MgSO₄ 0.50 g/l, FeSO₄ 0.01 g/l, agar 13.00 g/l) for 4 days, before being transferred to a sporulation medium (dextrin 2.27 g/l, urea 2.13 g/l, CaCl₂ 3.00 g/l, ZnSO₄·7H₂O 0.01 g/l, agar 13.00 g/l) for a further 4 days under the following environmental conditions: ?3.9 MPa/pH 7/light 24 h/temperature 29°C; these conditions were altered to ?0.3 MPa/pH 6/light 24 h/temperature 23°C in order to obtain better biomass yields. The data presented provide information on the nutrient and environmental requirements of this fungus, which will be essential for its commercial production.