World Journal of Microbiology and Biotechnology, 8 (Supplement 1), 79~82
Progress and prospects in ergot alkaloid research Z. I~eha.~ek and P. Sajdl Already known in the middle ages, ergot alkaloids (EAs) continue to be of great interest as drugs themselves or as prototype compounds from which new drugs are derived. EAs have been of prime interest to pharmacologists due to their multiple actions, which include effects on uterine and vascular muscle, and for their use as a tool for studying the mechanism of the sympathetic nervous system. Within the last few years new aspects of the pharmacology of EA derivatives have been recognized. These involve the action of certain EA derivatives as inhibitors of prolactin secretion, and stimulators of dopaminergic receptors. Thus, some EA derivatives have been implicated as potential therapeutic agents in many disease states such as Parkinsonism, acromegaly, amenorrhea-galactorrhea, suppression of postpar~um lactation, treatment of breast cancer, and possibly cancer of the prostate (secondary to inhibition of prolactin secretion). Innovation and technical development of EAs has moved closer to scientific research. Knowledge of physiological controls and genetic manipulation are dominant tools of modem EA production. This paper gives a brief review of recent knowledge in the physiological field of EA formation to illustrate the present problems and to emphasize areas where knowledge is lacking and where research should be persued further. Chemistry, biogenesis, biological activities and biotechnology of EAs have been extensively reviewed (Berde & Schild 1978, I~eha~ek 1980, 1986; l~eh~ek & Sajdl I990) and will not be described here.
Production of EAs In nature, EAs are formed primarily by the pyrenomycete fungus, Claviceps. However, EAs have also been found in the -Aspergillus and Penicillium genera, and in the Convolvu]aceae (morning-glory) family. Since the supply of natural-grown ergots is insufficient to meet current requirements, the The authors are at the Institute of Microbiology, Czechoslovak Academy of Sciences,. 142 20 Prague, Czechoslovakia.
biotechnological production of EAs has gained importance. 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. The history of the fermentation manufacture of EAs is basically a product of the filamentous fungus Claviceps. At present Claviceps may serve as an exceptionally useful tool for exploring the organization, expression and regulation of eukaryotic genomes. Remarkable progress has taken place in the description of enzymes involved in EA metabolism. This will be followed by cloning of the appropriate genes and the elucidation of primary structures of the alkaloids themselves. Structural genes and their control elements may then be manipulated to improve EA production and properties. The economic viability of EA production is determined to a large extent by its basic design and the extent to which its course can be controlled. More economic evaluation during the phase of innovation and the early stages of optimization and design is necessary, as well as the more intense collaboration of scientists belonging to the natural, technical, and economic faculties.
Enzymes of EA Metabolism
Claviceps possesses a great variety of enzymic activity and is versatile in its ability to utilize different compounds as growth substrates. It also has evolved efficient regulatory systems to ensure that the enzymes of a specific metabolic pathway are synthesized only when required and in proper amounts for efficient metabolism. EAs are generally produced by enzymes that are repressed or inhibited under conditions of rapid growth: EA synthesizing enzymes are present in mycelia grown in high levels of phosphate, are activated by decreasing the intracellular phosphate level, and do not undergo rapid turnover, in contrast to both alkaline and acid intracellular phosphatases (Pa~outov~ & l~ehfi~ek i984). The hypothesis appears to be that a common
1992 Rapid Communications of Oxford Ltd World Journal of Microbiology and Bios
Vol 8 Supplement I . 1992
79
Z. Rehdl~ek and P. Sajdl regulatory moment(s) may be participating in the synthesis of both alkaloids and lipids (Vo~i~ek & I~eh~ek 1978). Most enzymes catalysing different stages of EA metabolism are not known. Multiple enzymic complexes are involved in alkaloid metabolism and are not very stable. Work along these lines is in progress. Isopentenyl diphosphate isomerase (EC 5.3.3.2) (Satterwhite 1985) catalyses the reversible reaction providing dimethylallyl diphosphate, the 5-carbon allylic diphosphate subunit (isoprene!. Dimethylaltyltryptophan syntketase (Lee et al. 1976; Cress et al. 1981) catalyses the first reaction in a biosynthetic pathway of EAs in Claviceps sp. Dimethylallyltryptophan N-methyltransferase (Otsuka eta]. 1980) catalyses the transfer of the S-methyl group of S-adenosylmethionine to the amino group of 4-(3,3dimethylallyl) tryptophan. This methylation is the second pathway-specific step in ergoline biosynthesis. Chanoclavine-I cyclase (Gr6ger & Sajdl 1972; Erge et aI. 1973) characterizes a pathway from chanoclavine-I to elymoclavine. The requirement for FAD and NADP(H) indicates that an oxidation-reduction reaction is involved in the conversion of chanoclavine-I to agroclavine. Agroclavine hydroxylase (Hsu & Anderson 1971) catalyses the conversion of agroclavine to elymoclavine. Cytochrome P-450 (Rylko et aI. 1986) participates in the mono-oxygenation of N-methyldimethylallyltryptophan or 6, 7-secoagroclavine to chanoclavine-I, and chanoclavine-I to chanoclavine-I-aldehyde and it is involved in the conversion of agroclavine to elymoclavine and elymoclavine to paspalic acid. D-Lysergic acid-activating enzyme (Kim et al. 1983) catalyses both the D-lysergic acid-dependent ATP-pyrophosphate exchange and the formation of ATP from D-lysergic acid adenylate and pyrophosphate. Ergopeptine syntketase (Maier et al. 1983) catalyses the formation of the portion of the peptide part of ergopeptines.
Physiology of EA Formation Circumstantial evidence presently links the initiation of EA 9 metabolism to changes in a range of parameters: morphological; concentrations of enzymes and their substrates; nutrients and external stress. The biosynthesis of EAs lies at the level of genetic information apparatus and continues at the level of physiological realization (l~eh~ek 1983, 1990). Genes involved in EA synthesis are under rigorous metabolic control (Schmauder & Gr6ger 1976). The synthesis of the peptide portion of ergopeptines is a non-ribosomal process. Fungi and plants form ergolenes from the same precursors. EA formation is, to a greater degree, the result of changes in the physiological state rather than an indication of a new
~0
World Journal of Microbiology and Biotechnology, Vol 8 Supplement I 9 1992
physiological state (I~ehdt~ek 1972). Culture growth and EA synthesis are understood as processes which compete for key metabolic intermediates rather than as mutually exclusive phenomena. The biosynthesis of EAs requires a change in cell differentiation (Vo~ek et al. 1974: L6secke eta[. 1982). Differentiation and induction of EA synthesis start concomitantly in response to nutrient limitation. EAs are not waste, physiologically-inert, products of cell metabolism (I~ehdl~ek 1972). EA biosynthesis plays a role in the physiology o f the production organism by maintaining certain mechanisms of the metabolism and cell division in an operative state. The formation of clavine alkaloids is a part of endogenous metabolism releasing the energy required for maintenance of the integrity and viability of cells under conditions of limited growth (I~eh~ek eta]. 1973). In a productive cell, EAs increase the activity of tryptophan synthase and acetyl-CoA carboxylase, and inhibit anthranilate synthase, isocitrate lyase, malate synthase and ATPase (I~ehdl~eket al. 1971; Mann & Floss 1977). Agroclavine and elymoclavine inhibit purified dimethylallyltryptophan synthetase (Floss et aI. 1974; Floss 1976). In vitro studies (Erge et al. 1973) showed inhibition by elymoclavine of chanoclavine-I cyclase. Anthranilate synthetase is also inhibited by elymoclavine (Mann & Floss 1977). Ergometrine added to a submerged culture of C. paspali increases EA formation by 120%; ergotamine and ergometrine reduce the intensity of conidia formation in a production culture of C. purpurea (I~eh~ek et al. 1974).
Strain Improvement Improvement of industrial microorganisms has recently experienced intensive development attendant on the application of new genetic techniques and introduction of automated selection systems embodying robotized elements and computer technology. The discoveries of genetic modification, and other new methods such as cell fusion, provide far-reaching possibilities. Gene amplification in relation to EA production will be achieved in the next few years using molecular cloning techniques. From existing evidence it can be concluded that successful selection of deregulated mutants can be easily performed. The experimental data, far from being comprehensive, prove (Tudzynski et aI. 1983) that the concept to use mt plasmids or mtDNA as base to construct autonomously replicating vectors, is of general applicability. Naturally other extrachromosomal genetic traits may also be used in a comparable way for gene cloning. The occurrence of these elements, however, is as yet limited to a few organisms, whereas mtDNA is available from any eukaryotic organism. Strategies for the development of industrial strains must include considerations of the uptake and excretion of
Progress in ergot alkaloid research substrates and products that are not normally transported by the natural strain. The transmembrane transport processes are subject to regulation by cellular metabolism, and often are themselves primary regulators of metabolism. Membrane and membrane processes are likely to become a significant tool in the modern biotechnologist's armoury. The presence of virus might be expected to influence profoundly the biology and evolution of the Claviceps fungus. The relationship between viruses and fungal metabolism is at this time especially ill-defined, and virtually nothing is known about the genetic cycle of a fungal virus. Many cytoplasmically inherited characteristics of fungi may eventually prove to be viral in origin.
Future Prospects The future of EAs depends on a close interplay of microbiology, enzymology, genetics, chemistry, pharmacology, immunology and biochemical engineering. In the biosynthetic field of EAs, the mechanism and enzymology of formation of the C-ring, the D-ring and peptide moieties have yet to be worked out. Important findings concerning the physiology of EA formation could be provided by studies of membrane processes and vacuoles in productive organisms. Another topic of interest is the determination of the intracellular location of ergopeptine synthesis, and the distribution of alkaloids and their transport at the molecular level. According to recent investigations a concerted breeding of the ergot fungi by sexual (chromosomal) and parasexual (extrachromosomal) genetic engineering is now possible as a result of exploration of the molecular biology of EA metabolism and the availability of methods of recombinant DNA technology and reciprocal genetics. In the future, immobilization of multi-enzyme systems of EA formation can be developed if energy generation and oxidation-reduction reactions can be efficiently carried out. Coimmobilization of enzymes, cells and organelles from different organisms promises to improve further the industrial feasibility of immobilized biocatalysts. Mathematical modelling extended by physiological aspects could open a new area for a more exact and utilizable mathematical description of phenomena observed during culture growth and EA production. The range of EAs produced could be ,widened by development of new semisynthetic analogues that possess altered pharmacological effects. Rewarding research objectives would be to search for active drugs which are not subject to a first-pass effect, i.e. those which are not extensively metabolized in the liver, or substances which are handled by a deep compartment in some manner which enhances their therapeutic usefulness. In spite of past successes of EAs in practice, the future of these pharmaceuticals depends on the results of basic research.
References Berde, B. & Schild H.O. (Eds) 1978 Ergot Alkaloids and Related Compounds. Handbook of Experimental Pharmacology, Vol. 49. Berlin: Springer Verlag. Cress, W.A., Chayet, L.I. & Rilling H.C. 1981 Crystalization and partial characterization of dimethylallyl pyrophosphate: Ltryptophan dimethylallyltransferase from Claviceps sp. SD58. Journal of Biological Chemistry 256, 10917-10923. Erge, D., Maier, W. & Gr6ger, D. 1973 Enzymic conversion of chanoclavine-I. Biochem. Physiol. Pflanzen 164, 234-247. Floss, H.G., 1970 Biosynthesis of ergot alkaloids and related compounds. Tetrahedron 32, 873-912. Floss, H.G., Robbers, J.E. & Heinstein, P.F. 1974 Regulatory control mechanisms in alkaloid biosynthesis. Recent Advances in Phytochemistry 8, I41-178. Gr6ger, D. & SajdI P. 1972 Enzymatic conversion of chanocIavine-I Pharmazie 27, I88. Hsu J.C. & Anderson, J. A. 1971 Agroclavine hydroxylase of Claviceps purpurea. Biochimica et Biophysica Acta 230, 518-525. Kim, S.U., Cho, Y.J., Floss, H.G. & Anderson, J.A. 1983 Conversion of elymoclavine to paspalic acid by a particulate fraction from an ergotamine producing strain of Claviceps sp. Plata Med. 48, I45-148. Lee, S.D., Floss, H.G. & Heinstein P.F. 1976 Purification and properties of DMAPP tryptophan dimethylallyl transferase, the first enzyme of ergot biosynthesis in Claviceps sp. SD58. Archives of Biochemistry and Biophysics. 177, 84-94. L6secke, W., Neumann, D., Schmauder, H.P. & Gr6ger, D. I982 Changes in cytoplasmic ultrastructure during submerged cultivation of a peptide alkaloids-producing strain of Claviceps purpurea. Z Allg. Mikrobiol. 22, 49-61. Maier, W., Erge, D. & Gr6ger, D. I983 Further studies on cell-flee biosynthesis of ergotamine in Claviceps purpurea. FEMS Microbiology Letters. 20, 233-236. Mann, D.F., & Floss, H.G. 1977 Purification and properties of anthranilate synthetase from the ergot fungus Claviceps sp. strain SD58. Lloydia 40, 136-145. Otsuka, H., Quigley, F.R., Gr6ger, D., Anderson, J.A. & Floss, H.G. 1980 In vivo and in vitro evidence for N-methylation as the second pathway-specific step in ergoline biosynthesis. Planta Medica 40, 109-119. Pa~:outov~, S. & 15,eh~ek, Z. 1984 Phosphate regulation of phosphatases in submerged cultures of Claviceps purpurea 129 producing clavine alkaloids. Applied Microbiology and Biotechnofogy 20, 389-392. I~.eh~ek, Z. 1972 Physiology of formation of some antibiotics and ergot alkaloids (in Czech). DSc. Thesis, Institute of Microbiology, Czechoslovak Academy of Sciences, Prague. 15,ehfi~ek, Z. 1980 Ergot alkaloids and their biosynthesis. Advances in Biochemical Engineering, 14, 33-60. I~eh~ek, Z. 1983 New trends in ergot alkaloid biosynthesis. Proceedings of Biochemistry, 18, 22-33. I~eh~ek, Z. 1986 Formation physiology of ergot alkaloids. In Cell Metabolism: Growth and Environment, ed. Subramanian, T.A.V., Vol. II. pp. I01-I55. Boca Raton: CRC Press. I~eh~ek, Z. I990 Research into biogenesis of biologically active microbioal metabolites in Czechoslovakia: a review. Proceedings of Biochemistry 25, 197-209. I~eh~ek, Z. & Sajdl, P. 1990 Ergot Alkaloids: chemistry, biological effects, biotechnology. Amsterdam: Elsevier. I~eh~ek, Z., Kozova, J., Sajdl, P. & Ka~lik, J. 1973 Ergot alkaloid formation in relation to the cell-pool tryptophan and adenosine-5'-triphosphate. In Genetics of Industrial Microorganisms: actinomycetes and fungi, eds. Van6k, Z., Ho~f~lek, Z. & Cudlln, J., pp. 427-438. Amsterdam: Elsevier. World Journal of Microbiology and Biotechnology, VoI 8 Supplemenf I 9 1992
81
Z. Reh/~3ek and P. Saidl
I~eh~i~ek,Z., Kozova, J., Sajdl, P. & Vo~i~ek, J. 1974 The physiology of conidia formation in submerged culture of Chwiceps purl,urea (Fr.)Tul. producing alkaloids. Journal off Canadian Micn~l,iology 20, 1323-1329. 15,ehii~ek, Z., SajdI, P., Kozova, J., Malik, K.A. & t~i~icovii, A. 1971 Correlation of certain alterations in metabolic activity with the alkaloid production bY submerged Chwiceps cultures. Applied Microt,iolagy. 22, 949-956. Rylko, V., S~paI; Z., Sajdl, P. & t~eha~ek, Z. I980 Developmental profile of cytochrome P-450 in a submerged culture of the ergot fungus Chwiceps. 5th h#ernational Syntposium on Genetics of h~dustrial .Microorganisms. Abstr. p. I67, Split. Satterwhite, D.M. 1985 Metho& in Enzymology, eds. Law, J.H. &
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WorldJournalof Microbiologyand Biotechnology,Vol 8 Supplement 1 9 1992
Rilling, H.C., VoL 110, p. 92. New York: Academic Press. Schmauder, H.P. & Gr6ger, D. 1976 Zur Charakterizierung der Anthranilatsynthetase bei Claviceps. Biochem. Physiol. Pflanzen 169; 201-216. Tudzynski, P, Diivell, A. & Esser, K. 1983 Extrachromosomal genetics of Claviceps purpurea. L. Mitochondrial DNA and mitochondria[ plasmids. Curren~Genetics 7, 145-150. Vo?i~ek, J. & l~ehahek, Z. 1978 Fine structure localization of alkaloid synthesis in endoplasmic reticulum of submerged C. purpurea. Archives of Microbiology 117, 297-302. Vo/'i~ek; J., Ludvik, J. & l~eh~(:ek, Z. 1974 Morphogenesis and ultrastructure of C. purpurea during submerged alkaloid formation. Journal of Bacteriology. 120, 1401-1408.
Progress and prospects in ergot alkaloid research Z. I~eha.~ek and P. Sajdl Already known in the middle ages, ergot alkaloids (EAs) continue to be of great interest as drugs themselves or as prototype compounds from which new drugs are derived. EAs have been of prime interest to pharmacologists due to their multiple actions, which include effects on uterine and vascular muscle, and for their use as a tool for studying the mechanism of the sympathetic nervous system. Within the last few years new aspects of the pharmacology of EA derivatives have been recognized. These involve the action of certain EA derivatives as inhibitors of prolactin secretion, and stimulators of dopaminergic receptors. Thus, some EA derivatives have been implicated as potential therapeutic agents in many disease states such as Parkinsonism, acromegaly, amenorrhea-galactorrhea, suppression of postpar~um lactation, treatment of breast cancer, and possibly cancer of the prostate (secondary to inhibition of prolactin secretion). Innovation and technical development of EAs has moved closer to scientific research. Knowledge of physiological controls and genetic manipulation are dominant tools of modem EA production. This paper gives a brief review of recent knowledge in the physiological field of EA formation to illustrate the present problems and to emphasize areas where knowledge is lacking and where research should be persued further. Chemistry, biogenesis, biological activities and biotechnology of EAs have been extensively reviewed (Berde & Schild 1978, I~eha~ek 1980, 1986; l~eh~ek & Sajdl I990) and will not be described here.
Production of EAs In nature, EAs are formed primarily by the pyrenomycete fungus, Claviceps. However, EAs have also been found in the -Aspergillus and Penicillium genera, and in the Convolvu]aceae (morning-glory) family. Since the supply of natural-grown ergots is insufficient to meet current requirements, the The authors are at the Institute of Microbiology, Czechoslovak Academy of Sciences,. 142 20 Prague, Czechoslovakia.
biotechnological production of EAs has gained importance. 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. The history of the fermentation manufacture of EAs is basically a product of the filamentous fungus Claviceps. At present Claviceps may serve as an exceptionally useful tool for exploring the organization, expression and regulation of eukaryotic genomes. Remarkable progress has taken place in the description of enzymes involved in EA metabolism. This will be followed by cloning of the appropriate genes and the elucidation of primary structures of the alkaloids themselves. Structural genes and their control elements may then be manipulated to improve EA production and properties. The economic viability of EA production is determined to a large extent by its basic design and the extent to which its course can be controlled. More economic evaluation during the phase of innovation and the early stages of optimization and design is necessary, as well as the more intense collaboration of scientists belonging to the natural, technical, and economic faculties.
Enzymes of EA Metabolism
Claviceps possesses a great variety of enzymic activity and is versatile in its ability to utilize different compounds as growth substrates. It also has evolved efficient regulatory systems to ensure that the enzymes of a specific metabolic pathway are synthesized only when required and in proper amounts for efficient metabolism. EAs are generally produced by enzymes that are repressed or inhibited under conditions of rapid growth: EA synthesizing enzymes are present in mycelia grown in high levels of phosphate, are activated by decreasing the intracellular phosphate level, and do not undergo rapid turnover, in contrast to both alkaline and acid intracellular phosphatases (Pa~outov~ & l~ehfi~ek i984). The hypothesis appears to be that a common
1992 Rapid Communications of Oxford Ltd World Journal of Microbiology and Bios
Vol 8 Supplement I . 1992
79
Z. Rehdl~ek and P. Sajdl regulatory moment(s) may be participating in the synthesis of both alkaloids and lipids (Vo~i~ek & I~eh~ek 1978). Most enzymes catalysing different stages of EA metabolism are not known. Multiple enzymic complexes are involved in alkaloid metabolism and are not very stable. Work along these lines is in progress. Isopentenyl diphosphate isomerase (EC 5.3.3.2) (Satterwhite 1985) catalyses the reversible reaction providing dimethylallyl diphosphate, the 5-carbon allylic diphosphate subunit (isoprene!. Dimethylaltyltryptophan syntketase (Lee et al. 1976; Cress et al. 1981) catalyses the first reaction in a biosynthetic pathway of EAs in Claviceps sp. Dimethylallyltryptophan N-methyltransferase (Otsuka eta]. 1980) catalyses the transfer of the S-methyl group of S-adenosylmethionine to the amino group of 4-(3,3dimethylallyl) tryptophan. This methylation is the second pathway-specific step in ergoline biosynthesis. Chanoclavine-I cyclase (Gr6ger & Sajdl 1972; Erge et aI. 1973) characterizes a pathway from chanoclavine-I to elymoclavine. The requirement for FAD and NADP(H) indicates that an oxidation-reduction reaction is involved in the conversion of chanoclavine-I to agroclavine. Agroclavine hydroxylase (Hsu & Anderson 1971) catalyses the conversion of agroclavine to elymoclavine. Cytochrome P-450 (Rylko et aI. 1986) participates in the mono-oxygenation of N-methyldimethylallyltryptophan or 6, 7-secoagroclavine to chanoclavine-I, and chanoclavine-I to chanoclavine-I-aldehyde and it is involved in the conversion of agroclavine to elymoclavine and elymoclavine to paspalic acid. D-Lysergic acid-activating enzyme (Kim et al. 1983) catalyses both the D-lysergic acid-dependent ATP-pyrophosphate exchange and the formation of ATP from D-lysergic acid adenylate and pyrophosphate. Ergopeptine syntketase (Maier et al. 1983) catalyses the formation of the portion of the peptide part of ergopeptines.
Physiology of EA Formation Circumstantial evidence presently links the initiation of EA 9 metabolism to changes in a range of parameters: morphological; concentrations of enzymes and their substrates; nutrients and external stress. The biosynthesis of EAs lies at the level of genetic information apparatus and continues at the level of physiological realization (l~eh~ek 1983, 1990). Genes involved in EA synthesis are under rigorous metabolic control (Schmauder & Gr6ger 1976). The synthesis of the peptide portion of ergopeptines is a non-ribosomal process. Fungi and plants form ergolenes from the same precursors. EA formation is, to a greater degree, the result of changes in the physiological state rather than an indication of a new
~0
World Journal of Microbiology and Biotechnology, Vol 8 Supplement I 9 1992
physiological state (I~ehdt~ek 1972). Culture growth and EA synthesis are understood as processes which compete for key metabolic intermediates rather than as mutually exclusive phenomena. The biosynthesis of EAs requires a change in cell differentiation (Vo~ek et al. 1974: L6secke eta[. 1982). Differentiation and induction of EA synthesis start concomitantly in response to nutrient limitation. EAs are not waste, physiologically-inert, products of cell metabolism (I~ehdl~ek 1972). EA biosynthesis plays a role in the physiology o f the production organism by maintaining certain mechanisms of the metabolism and cell division in an operative state. The formation of clavine alkaloids is a part of endogenous metabolism releasing the energy required for maintenance of the integrity and viability of cells under conditions of limited growth (I~eh~ek eta]. 1973). In a productive cell, EAs increase the activity of tryptophan synthase and acetyl-CoA carboxylase, and inhibit anthranilate synthase, isocitrate lyase, malate synthase and ATPase (I~ehdl~eket al. 1971; Mann & Floss 1977). Agroclavine and elymoclavine inhibit purified dimethylallyltryptophan synthetase (Floss et aI. 1974; Floss 1976). In vitro studies (Erge et al. 1973) showed inhibition by elymoclavine of chanoclavine-I cyclase. Anthranilate synthetase is also inhibited by elymoclavine (Mann & Floss 1977). Ergometrine added to a submerged culture of C. paspali increases EA formation by 120%; ergotamine and ergometrine reduce the intensity of conidia formation in a production culture of C. purpurea (I~eh~ek et al. 1974).
Strain Improvement Improvement of industrial microorganisms has recently experienced intensive development attendant on the application of new genetic techniques and introduction of automated selection systems embodying robotized elements and computer technology. The discoveries of genetic modification, and other new methods such as cell fusion, provide far-reaching possibilities. Gene amplification in relation to EA production will be achieved in the next few years using molecular cloning techniques. From existing evidence it can be concluded that successful selection of deregulated mutants can be easily performed. The experimental data, far from being comprehensive, prove (Tudzynski et aI. 1983) that the concept to use mt plasmids or mtDNA as base to construct autonomously replicating vectors, is of general applicability. Naturally other extrachromosomal genetic traits may also be used in a comparable way for gene cloning. The occurrence of these elements, however, is as yet limited to a few organisms, whereas mtDNA is available from any eukaryotic organism. Strategies for the development of industrial strains must include considerations of the uptake and excretion of
Progress in ergot alkaloid research substrates and products that are not normally transported by the natural strain. The transmembrane transport processes are subject to regulation by cellular metabolism, and often are themselves primary regulators of metabolism. Membrane and membrane processes are likely to become a significant tool in the modern biotechnologist's armoury. The presence of virus might be expected to influence profoundly the biology and evolution of the Claviceps fungus. The relationship between viruses and fungal metabolism is at this time especially ill-defined, and virtually nothing is known about the genetic cycle of a fungal virus. Many cytoplasmically inherited characteristics of fungi may eventually prove to be viral in origin.
Future Prospects The future of EAs depends on a close interplay of microbiology, enzymology, genetics, chemistry, pharmacology, immunology and biochemical engineering. In the biosynthetic field of EAs, the mechanism and enzymology of formation of the C-ring, the D-ring and peptide moieties have yet to be worked out. Important findings concerning the physiology of EA formation could be provided by studies of membrane processes and vacuoles in productive organisms. Another topic of interest is the determination of the intracellular location of ergopeptine synthesis, and the distribution of alkaloids and their transport at the molecular level. According to recent investigations a concerted breeding of the ergot fungi by sexual (chromosomal) and parasexual (extrachromosomal) genetic engineering is now possible as a result of exploration of the molecular biology of EA metabolism and the availability of methods of recombinant DNA technology and reciprocal genetics. In the future, immobilization of multi-enzyme systems of EA formation can be developed if energy generation and oxidation-reduction reactions can be efficiently carried out. Coimmobilization of enzymes, cells and organelles from different organisms promises to improve further the industrial feasibility of immobilized biocatalysts. Mathematical modelling extended by physiological aspects could open a new area for a more exact and utilizable mathematical description of phenomena observed during culture growth and EA production. The range of EAs produced could be ,widened by development of new semisynthetic analogues that possess altered pharmacological effects. Rewarding research objectives would be to search for active drugs which are not subject to a first-pass effect, i.e. those which are not extensively metabolized in the liver, or substances which are handled by a deep compartment in some manner which enhances their therapeutic usefulness. In spite of past successes of EAs in practice, the future of these pharmaceuticals depends on the results of basic research.
References Berde, B. & Schild H.O. (Eds) 1978 Ergot Alkaloids and Related Compounds. Handbook of Experimental Pharmacology, Vol. 49. Berlin: Springer Verlag. Cress, W.A., Chayet, L.I. & Rilling H.C. 1981 Crystalization and partial characterization of dimethylallyl pyrophosphate: Ltryptophan dimethylallyltransferase from Claviceps sp. SD58. Journal of Biological Chemistry 256, 10917-10923. Erge, D., Maier, W. & Gr6ger, D. 1973 Enzymic conversion of chanoclavine-I. Biochem. Physiol. Pflanzen 164, 234-247. Floss, H.G., 1970 Biosynthesis of ergot alkaloids and related compounds. Tetrahedron 32, 873-912. Floss, H.G., Robbers, J.E. & Heinstein, P.F. 1974 Regulatory control mechanisms in alkaloid biosynthesis. Recent Advances in Phytochemistry 8, I41-178. Gr6ger, D. & SajdI P. 1972 Enzymatic conversion of chanocIavine-I Pharmazie 27, I88. Hsu J.C. & Anderson, J. A. 1971 Agroclavine hydroxylase of Claviceps purpurea. Biochimica et Biophysica Acta 230, 518-525. Kim, S.U., Cho, Y.J., Floss, H.G. & Anderson, J.A. 1983 Conversion of elymoclavine to paspalic acid by a particulate fraction from an ergotamine producing strain of Claviceps sp. Plata Med. 48, I45-148. Lee, S.D., Floss, H.G. & Heinstein P.F. 1976 Purification and properties of DMAPP tryptophan dimethylallyl transferase, the first enzyme of ergot biosynthesis in Claviceps sp. SD58. Archives of Biochemistry and Biophysics. 177, 84-94. L6secke, W., Neumann, D., Schmauder, H.P. & Gr6ger, D. I982 Changes in cytoplasmic ultrastructure during submerged cultivation of a peptide alkaloids-producing strain of Claviceps purpurea. Z Allg. Mikrobiol. 22, 49-61. Maier, W., Erge, D. & Gr6ger, D. I983 Further studies on cell-flee biosynthesis of ergotamine in Claviceps purpurea. FEMS Microbiology Letters. 20, 233-236. Mann, D.F., & Floss, H.G. 1977 Purification and properties of anthranilate synthetase from the ergot fungus Claviceps sp. strain SD58. Lloydia 40, 136-145. Otsuka, H., Quigley, F.R., Gr6ger, D., Anderson, J.A. & Floss, H.G. 1980 In vivo and in vitro evidence for N-methylation as the second pathway-specific step in ergoline biosynthesis. Planta Medica 40, 109-119. Pa~:outov~, S. & 15,eh~ek, Z. 1984 Phosphate regulation of phosphatases in submerged cultures of Claviceps purpurea 129 producing clavine alkaloids. Applied Microbiology and Biotechnofogy 20, 389-392. I~.eh~ek, Z. 1972 Physiology of formation of some antibiotics and ergot alkaloids (in Czech). DSc. Thesis, Institute of Microbiology, Czechoslovak Academy of Sciences, Prague. 15,ehfi~ek, Z. 1980 Ergot alkaloids and their biosynthesis. Advances in Biochemical Engineering, 14, 33-60. I~eh~ek, Z. 1983 New trends in ergot alkaloid biosynthesis. Proceedings of Biochemistry, 18, 22-33. I~eh~ek, Z. 1986 Formation physiology of ergot alkaloids. In Cell Metabolism: Growth and Environment, ed. Subramanian, T.A.V., Vol. II. pp. I01-I55. Boca Raton: CRC Press. I~eh~ek, Z. I990 Research into biogenesis of biologically active microbioal metabolites in Czechoslovakia: a review. Proceedings of Biochemistry 25, 197-209. I~eh~ek, Z. & Sajdl, P. 1990 Ergot Alkaloids: chemistry, biological effects, biotechnology. Amsterdam: Elsevier. I~eh~ek, Z., Kozova, J., Sajdl, P. & Ka~lik, J. 1973 Ergot alkaloid formation in relation to the cell-pool tryptophan and adenosine-5'-triphosphate. In Genetics of Industrial Microorganisms: actinomycetes and fungi, eds. Van6k, Z., Ho~f~lek, Z. & Cudlln, J., pp. 427-438. Amsterdam: Elsevier. World Journal of Microbiology and Biotechnology, VoI 8 Supplemenf I 9 1992
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Z. Reh/~3ek and P. Saidl
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