Selection of ergot alkaloid producers by induced mutagenesis

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

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.     

Physiological control and process kinetics of clavine alkaloid production byClaviceps purpurea

Summary Kinetic parameters of production of clavine alkaloids were evaluated in twoClaviceps purpurea strains. Mutagenesis brought about enhanced resistance of the biosynthetic system towards alkaloids. Addition of glucose into the fermentation medium altered the zero order kinetics of production to activation-inhibition kinetics. The glucose treatment allowed performance of both elymoclavine-inhibitionless and clavine alkaloid-decompositionless fermentations if a combination of fermentation and separation units in a closed loop was used.Nomenlacture k ₁ rate constant of agroclavine synthesis (mg Agro · mg Elymo/l·g DW·day for stage 1, mg Agro/g DW·day for stage 2) - k ₂ parameter describing inhibition of agroclavine formation rate by elymoclavine (mg Elymo/l) - k ₃ specific rate of agroclavine decay (l/g DW·day) - k ₄ maximal specific rate of elymoclavine synthesis (stage 1, 1/g DW·day, stage 2, mg Elymo/g DW·day) - k ₄ ^– maximal specific rate of elymoclavine synthesis in stage 1 (inhibition-activation mechanism) (mg Elymo/g DW·day) - k ₅ physiological constant describing the elymoclavine decay rate (l²/g DW·day·mg Elymo) - k ₅ ^– physiological constant describing the activation of elymoclavine biosynthesis by elymoclavine (mg Elymo/l) - k ₆ physiological constant describing the repression of elymoclavine biosynthesis by elymoclavine (mg Elymo/l) - k ₇ maximal specific growth rate (1/day) - k ₈ specific rate of biomass decay (l/g DW·day) - A agroclavine concentration (mg/l) - E elymoclavine concentration (mg/l) - r _A specific rate of agroclavine biosynthesis (mg Agro/g DW·day) - r _E specific rate of elymoclavine biosynthesis (mg Elymo/g DW·day) - r _i specific rate of alkaloid biosynthesis (mg alkaloid/g DW·day) - X dry biomass concentration (g/l) - specific growth rate (1/day) Abbreviations Agro agroclavine - Elymo elymoclavine - Chano chanoclavine - DW dry weight of biomass     

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 c106 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°C in T25 supplemented by 7% glycerol provided a stable level of alkaloid formation.     

hREV3 is essential for error-prone translesion synthesis past UV or benzo[a]pyrene diol epoxide-induced DNA lesions in human fibroblasts

In S. cerevisiae, the REV3 gene, encoding the catalytic subunit of polymerase zeta, is involved in translesion synthesis and required for the production of mutations induced by ultraviolet radiation (UV) photoproducts and other DNA fork-blocking lesions, and for the majority of spontaneous mutations. To determine whether hREV3, the human homolog of yeast REV3, is similarly involved in error-prone translesion synthesis past UV photoproducts and other lesions that block DNA replication, an hREV3 antisense construct under the control of the TetP promoter was transfected into an infinite life span human fibroblast cell strain that expresses a high level of tTAk, the activator of that promoter. Three transfectant strains expressing high levels of hREV3 antisense RNA were identified and compared with their parental cell strain for sensitivity to the cytotoxic and mutagenic effects of UV. The three hREV3 antisense-expressing cell strains were not more sensitive than the parental strain to the cytotoxic effect of UV, but the frequency of mutants induced by UV in their HPRT gene was significantly reduced, i.e. to 14% that of the parent. Two of these hREV3 antisense-expressing cell strains were compared with the parental strain for sensitivity to (±)-7β,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). They were not more sensitive than the parent strain to the cytotoxic effect of BPDE, but the frequency of mutants induced was significantly reduced, i.e. in one strain, to 17% that of the parent, and in the other, to 24%. DNA sequencing showed that the kinds of mutations induced by BPDE in the parental and the derivative strains did not differ and were similar to those found previously with finite life span human fibroblasts. The data strongly support the hypothesis that hRev3 plays a critical role in the induction of mutations by UV or BPDE. Because the level of hRev3 protein in human fibroblasts is below the level of antibody detection, it was not possible to demonstrate that the decrease in mutagenesis reflected decreased hRev3 protein. However, the conclusion is supported by the fact that in a similar study with a strain expressing a high level of antisense hREV1, a very similar result was obtained, i.e. UV or BPDE mutagenesis was virtually eliminated.     

Isolation and characterization of pigment mutants from a filamentous cyanobacterium

Five strains of a pigment mutant were isolated following UV irradiation and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) mutagenesis from a non-nitrogen fixing mutant of the cyanobacteriumGloeotrichia ghosei. Two of them (B-1 and V-1) were isolated by UV mutagenesis and other three (B-3, B-7 and Br-6) by MNNG mutagenesis. Among the five strains cultures of three strains (B-1, B-3 and B-7) were typically blue-green in colour. Culture of strain V-1 was found to be violet-pink and of Br-6 was brownish in colour. The parent strain of these mutants was dark-blue in colour. Blue-green mutants showed the predominance of phycocyanin (610 nm) whereas violet-pink and brown strains showed the predominance of phycoerythrin (550 nm) in the absorption spectra of water-soluble pigments. In contrast to these strains their parent strain showed both the absorption peaks (at 550 and 610 nm). Occurrence of stable pigment mutants of a filamentous cyanobacterium indicates that the synthesis of water-soluble pigments is genetically controlled in these mutant strains.     

Inducible character of β‐xylanase in a hyperproducing mutant of Thermomyces lanuginosus

Ten strains of Thermomyces lanuginosus from various culture collections were evaluated for extracellular endo‐β‐1,4‐xylanase production. The best xylanase producer (5771±173 nkat/mL) T. lanuginosus SK, was subjected to UV and N‐methyl‐N‐nitro‐N‐nitrosoguanidine mutagenesis. A mutant strain T. lanuginosus MC134, that showed on oatspelts xylan a 1.5 fold higher xylanase production than the parent strain SK, was subjected to a study of the regulation of xylanase synthesis during growth on various carbohydrates and during induction in glucose‐grown cells. In the growth experiments the highest production of xylanase was observed in the presence of xylans, however, an appreciable amount of the enzyme, about 10%, was also produced during growth on xylose. Xylobiose was found to be the most efficient xylanase inducer in the glucose‐grown cells. Its induction efficiency was followed by xylose, beechwood and birchwood xylan. Xylanase induction by polysaccharides started several hours later but proceeded for a longer time than that induced by the low molecular mass inducers, indicating that the polysaccharides serve as more sustainable source of inducers and that they have to be first hydrolyzed by the low level of constitutively synthesized xylanase. The repression of the induction of xylanase by glucose confirmed that the xylanase synthesis in the mutant strain is similar to the parent strain and exhibits an induction‐repression regulation mechanism.     

A new Penicillium echinulatum strain with faster cellulase secretion obtained using hydrogen peroxide mutagenesis and screening with 2-deoxyglucose

Aims: The aim of this study is to improve cellulase production and secretion by Penicillium echinulatum using mutagenesis and selection in association with microfermentation and microanalysis methods. Methods and Results: A new genetic variant was isolated from strain 9A02S1 and named S1M29. It was obtained by mutagenesis with H₂O₂ and two screening steps, which involved selection in Petri dishes using the medium supplemented with 2‐deoxyglucose and microfermentations in submerged culture. The mutant showed higher cellulase productivity than 9A02S1 based on the Filter Paper Activity assay and endoglucanase; the peak activities for these enzymes were reached significantly faster than for the parent strain. Conclusions: The mutant obtained after mutagenesis and selection could produce and secrete cellulase faster than the parent strain. Significance and Impact of the Study: Mutagenesis followed by selection is a useful tool for rapidly generating new cellulase‐producing phenotypes in fungi. Faster production and higher titers of cellulases in mutant strains contribute to reduce the production costs for enzymatic complexes that hydrolyse lignocellulose residues and form fermented syrups, thus contributing to the economic production of bioethanol.     

Screening and selection of exopolysaccharide-producing strains of Lactobacillus delbrueckii subsp. bulgaricus

AIMS: The selection of exopolysaccharide (EPS)-producing strains of Lactobacillus delbrueckii subsp. bulgaricus. METHODS AND RESULTS: Improved EPS-overproducing strains of L. delbrueckii subsp. bulgaricus were derived by chemical mutagenesis and selection. Initial screening of the chemically induced mutant pool relied primarily on the selection of strains with raised levels of lactic acid and reduced biomass formation. Supporting selection criteria used were ropiness and colonial mucoidy. Final screening of candidate strains undertaken in a semi-defined medium in batch culture, resulted in the selection of a mutant with a 35% improvement in specific EPS yield relative to the parent strain. CONCLUSIONS: Initial selection of mutants of L. delbrueckii subsp. bulgaricus on the basis of enhanced formation of lactate and reduced biomass formation, coupled with a ropy or mucoid phenotype, proved to be a satisfactory means of isolating strains with the potential for a higher level of specific EPS production than the parent strain. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay protocol allowed for the selection of an EPS-overproducing strain of L. delbrueckii subsp. bulgaricus. Such strains are useful for the purposes of metabolic studies related to EPS-production.     

Synthesis and Antiviral Evaluation of N-β-D-Ribosides of Ergot Alkaloids

Abstract

N-β-D-Ribosides of agroclavine (1), elymoclavine (2), lysergene (4), lysergol (3), and 9, 10-dihydrolysergol (5) were prepared by SnCl₄ catalyzed ribosylation of their TMS derivatives with 1-O-acetyl-2, 3,5-tri-O-benzoyl-β-D-ribofuranose. None of the new compounds exhibited activity against HIV or other viruses tested.     

Interstrain spheroplast fusion of Alternaria alternata

Summary Auxotrophic and drug resistant colonies of Alternaria alternata were selected following UV mutagenesis of spheroplasts and genetic transformation with pDH25. Intrastrain cell fusion of certain A. alternata parental strains induced by polyethylene glycol occurred at an average rate of 0.35%; interstrain fusions occurred at a rate of 0.08%. Mitotic recombination resulted from UV mutagenesis of spheroplasts from several fusants from 6hy1 × 1ar1. Fusants synthesized different levels of the cyclic tetrapeptide, tentoxin; some colonies produced higher levels than either parent. These results demonstrate that spheroplast fusion may have a potential application for genetic analysis of secondary metabolite production and for strain improvement in A. alternata.Mention of a trademark of proprietary product does not constitute a guarantee or warranty by the U. S. Department of Agriculture and does not imply approval to the exclusion of other products that may also be suitable.     

Synthesis of different pectinases by filamentous growingA. niger mutants

Mutants ofA. niger K 69/26, prepared by multistep mutagenesis (UV, MNNG, heating) have been screened for pectinase activities. Mutants with altered levels of certain pectinases, such as endo- and exopolygalacturonase (PG vis, red), pectinesterase (PE) and pectinlyase (PL), were isolated. The enzyme activities of the best mutants M 1348/126 were increased 2–3-fold compared to the parent strain after a 6-d cultivation of filamentous mycelium on a shaker. Further mutagenesis of mutants with decreased pectinase activities (e.g. Se3) produced revertants. PG (vis) synthesis of revertant Se5 was increased 1.7 times compared to the control strain K 69/26. Independent of these increased rates, the general level of pectinase activities synthesized by the filamentous mycelium ofA. niger mutants amounts to about 10–20% compared with those produced by aggregated mycelium. It appears that the enzyme synthesis related to mycelium structure is independent of the mechanism which regulates the level of pectinase synthesis within a specific morphological structure.     

Effect of cultivation temperature,clomiphene, and nystatin on the oxidation and cyclization of chanoclavine in submerged cultures of the mutant strainclaviceps purpurea 59

Submerged cultures ofClaviceps purpurea 59 produce predominantly tricyclic chanoclavine and chanoclavine-I-aldehyde; formation of the tetracyclic agroclavine and elymoclavine is limited to 20%. The production cultures were characterized by the oxidation index o_i (100% chanoclavine/% chanoclavine) and cyclization index c_i (% agroclavine +% elymoclavine/% chanoclavine-I-aldehyde), expressing two functions of chanoclavine cyclase. Both indices were influenced by cultivation temperature and membrane agents. At 20°–24°C, clomiphene increased o_i and c_i; at 28°C and more it increased o_i only. At 24°C nystatin increased o_i and decreased c_i. During cultivation of vegetative inocula and production cultures at various temperatures (24°, 28°, and 33°C), the lower cultivation temperature of the inoculum increased c_i of the production culture with the higher temperature. The higher cultivation temperature of the inoculum decreased o_i and particularly c_i of the production culture with the lower temperature. In production cultures cultivated from the inoculum of an identical temperature, o_i decreased to 50% with increasing temperature above 24°C, whereas c_i decreased continuously. The role of the chanoclavine cyclase conformation as a membrane enzyme is discussed.     

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.     

ENHANCED β-GALACTOSIDASE PRODUCTION OF Aspergillus oryzae MUTATED BY UV AND LiCl

In order to breed a high-yield β-galactosidase-producing strain, Aspergillus oryzae was used as the parent strain and mutagenized with ultraviolet (UV) and UV plus lithium chloride (LiCl), respectively. After being mutagenized by UV, the β-galactosidase activity of mutant UV-15-20 reached 114.08 U/mL, which revealed a 49.22% increase compared with the original strain. A mutant UV-LiCl-38 with high β-galactosidase activity (121.42U/mL) was obtained after compound mutagenesis of UV and LiCl; the β-galactosidase activity of this mutant was 58.82% higher than that of the parent strain. Subculture testing indicated that UV-15-20 and UV-LiCl-38 had good hereditary stability and may be ideal strains for the production of β-galactosidase. Additionally, it was demonstrated that compound mutagenesis with UV and LiCl is an effective mutation method for breeding industrially interesting strains.     

Effects of clavines, ergot alkaloids, on the membrane potential of an identifiable giant neuron of the African giant snail Achatina fulica Ferussac

The effects of seven clavines, alkaloids of ergot, on the electrical activity of an identifiable giant neurone (TAN, tonically autoactive neurone) of the African giant snail were examined. All the substances examined, lysergine, agroclavine, elymoclavine, festuclavine, chanoclavine, rugulovasine A and rugulovasine B, at 2 X 10(-4) kg/l have no constant effect on TAN, indicating that they have no direct effect on this neurone. However, the substances examined, except for chanoclavine, in the same concentration occasionally caused the transient depression with an augmentation of trans-synaptic influences. This depression may be due to the trans-synaptic influences. The four substances examined, lysergine, agroclavine, elymoclavine and festuclavine, in the same concentration produced TAN abnormal spike discharges, doublet or triplet spikes.     

Properties of heterozygous diploids between strains ofPenicillium chrysogenum selected for high penicillin yield

Three strains ofPenicillium chrysogenum selected for high penicillin yield and of independent lineage were marked with suitable genetical characters prior to the synthesis of several heterozygous diploids. These parental strains had domestic codes, C, D and Y. Two diploids, between differently labelled mutants of strain C and Y, produced similar amounts of penicillin to strain C, which was less than that produced by strain Y. Previous work had indicated that genes responsible for increased penicillin yield were recessive and the present results suggested that such genes in strains C and Y were allelic, apart from the presence of one or more additional recessive mutations leading to greater penicillin production in the higher yielding parent. Three diploids made between mutants of strains D and Y were lower in penicillin yield than either original parent and only in the case of one diploid compared with one of the parental strains was this difference not significant. In strains D and Y, therefore, there may have been some recessive genes concerned with increasing penicillin yield which were non-allelic. However, no first order segregants arising spontaneously or subsequent to X-ray treatment produced higher levels of penicillin than the better yielding original parent in any cross.     

Separation of ergot alkaloids by adsorption on silicates

A mixture of ergot alkaloids (agroclavine, elymoclavine, chanoclavine, and chanoclavine aldehyde) was separated from the Claviceps purpureafermentation broth by adsorption on inorganic adsorbents containing silica. The uptake of alkaloids depended on the concentration of adsorbent and pH. The adsorption capacity for of inorganic materials increased with increasing content of inorganic oxides such as MgO and CaO in the adsorbent. Using statistical thermodynamics, a simple mathematical model describing the multicomponent adsorption equilibrium is proposed and a numerical method suitable for fast computer simulation of multicomponent adsorption was developed.     

Glycosylation of ergot alkaloids by free and immobilized cells of Claviceps purpurea

Summary A new strain, Claviceps purpurea 88-EP-47, with high invertase activity was selected. Free and Calginate immobilized cultures of this strain were used for fructosylation of ergot alkaloids. By bioconversion from their aglycones, elymoclavine-O--d-fructofuranosyl(21)-O--d-fructofuranoside, and elymoclavine-O--d-fructoside, the respective fructosides of chanoclavine, lysergol and dihydro-lysergol monofructosides were obtained. These substances are formed by -d-fructofuranosidase present in Claviceps cells. The bioconversion activity of the enzyme system (fructose transfer) is strongly dependent on pH, substrate (sucrose) concentration and the developmental profile of invertase activity. The pH optimum for elymoclavine fructosylation is 6.5, for chanoclavine 5.7, and the optimal sucrose concentration is 75 g/l. Fifteen-day-old production cultures had the best glycosylation activities. Fructosylation of alkaloids can be stimulated in production cultures of C. purpurea or C. fusiformis forming elymoclavine or chanoclavine by a pH shift to 6.5 at the end of the production phase. Glycosylating Claviceps strains producing elymoclavine eliminate the free alkaloid into glycosides. The feedback inhibition of alkaloid synthesis by elymoclavine is then strongly reduced, helping to further improve elymoclavine yields. Elymoclavine can be liberated simply by invertase activity of baker's yeast.Offprint requests to: V. Ken     

Mitogenic Effect of the Mannans from Saccharomyces cerevisiae on Mouse Spleen Lymphocytes

The DNA synthetic activities of mannans isolated from two Saccharomyces cerevisiae strains were examined in vitro using spleen cells obtained from normal or nude BALB/c strain mice. A highly branched mannan isolated from the S. cerevisiae wild type strain induced a greater increase in mitogenic activity than those displayed by the mannan of the S. cerevisiae X2180–1A-5 mutant strain which possessed fewer branching moieties. Acid-hydrolyzed wild type strain mannan with two-thirds of the molecular weight of the parent intact mannan showed weak mitogenicity. Increases in the DNA synthetic activities of nude and normal spleen cells were almost the same as that of wild type strain mannan, while nylon wool column-passed spleen cells obtained from both normal and nude mice did not show mitogenicity with this mannan. The results indicated that the mitogenic activity was responsible for the highly branched structure of the wild type strain mannan, and that this mannan is a B-cell mitogen.