Tolypocladium Inflatum Research Articles

The peptide synthetase catalyzing cyclosporine production in Tolypocladium niveum is encoded by a giant 45.8-kilobase open reading frame

Cyclosporin A, a potent and clinically-important immunosuppressive drug (SandimmunR), is synthesized from its precursor amino acids by cyclosporin synthetase, a single multi-functional enzyme. In this study we report the cloning of the corresponding coding region of this synthetase. It contains an open reading frame of 45.8 kb which encodes a peptide with a calculated M(r) of 1,689,243. The predicted gene product contains 11 amino-acid-activating domains that are very similar to one another and to the domains of other peptide synthetases. Seven of these domains harbour N-methyltransferase functions. This is the largest genomic ORF described so far.

Purification and characterization of eucaryotic alanine racemase acting as key enzyme in cyclosporin biosynthesis.

A specific alanine racemase, which is a key enzyme in the biosynthesis of the undecapeptide cyclosporin A, was purified to electrophoretic homogeneity from the fungus Tolypocladium niveum. This is the first enzyme of this kind isolated from a eucaryotic organism. The enzyme catalyzes the reversible racemization of alanine and requires pyridoxal phosphate as the exclusive cofactor. Km values for L- and D-alanine were found to be 38 and 2 mM, respectively. Maximal reaction velocity was observed at 42 degrees C and pH 8.8 for the L to D direction. Molecular mass determinations of the denatured enzyme by SDS-polyacrylamide gel electrophoresis gave a value of 37 kDa, whereas gel filtration calibration studies yielded a value between 120 and 150 kDa, indicating an oligomeric native structure.

Disruption of the cyclosporin synthetase gene of Tolypocladium niveum.

Cyclosporin A is a potent and clinically-important immunosuppressive drug (SandimmunR). It is produced by the fungus Tolypocladium niveum. A transformation system for T. niveum ATCC34921 based on hygromycin selection was established. In order to obtain a T. niveum promoter, the cyclophilin gene was isolated using the Neurospora crassa gene as probe. A plasmid vector was constructed in which the promoter region of the T. niveum cyclophilin gene was fused to a bacterial hygromycin phosphotransferase gene. Protoplasts were transformed with this plasmid and hygromycin-resistant transformants were isolated. Using this transformation system, mutants of T. niveum with disrupted versions of the cyclosporin synthetase gene (simA) were engineered by DNA-mediated transformation. Disruption of the gene resulted in loss of the ability to produce cyclosporins.

Biosynthesis of the unusual amino acid (4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine of cyclosporin A. Identification of 3(R)-hydroxy-4(R)-methyl-6(E)-octenoic acid as a key intermediate by enzymatic in vitro synthesis and by in vivo labeling techniques.

The biosynthesis of (4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine (abbreviation: Bmt, systematic name: 2(S)-amino-3(R)-hydroxy-4(R)-methyl-6(E)-octenoic acid) is proposed to involve two principal phases: the formation of a polyketide backbone and a subsequent transformation process to the final product. Here we report on the identification of 3(R)-hydroxy-4(R)-methyl-6(E)-octenoic acid as the end product of the first phase. The primary indication of 3(R)-hydroxy-4(R)-methyl-6(E)-octenoic acid as the key intermediate in the proposed biosynthetic route came from in vivo labeling studies with [1-13C,18O2]acetate, demonstrating retention of 18O in the 3-hydroxy group. Final identification of this intermediate in in vitro polyketide assays with enriched enzyme fractions of Tolypocladium niveum was achieved after development of highly sensitive and specific detection methods and by use of synthetic reference substances. Two additional methylated in vitro products could be detected and characterized as 4(R)-methyl-(E,E)-2,6-octadienoic acid and 4(R)-methyl-6(E)-octenoic acid by liquid chromatography-mass spectrometry analysis and comparison with synthetic reference samples. Their relevance for Bmt biosynthesis is discussed. Bmt polyketide synthase shows optimal activity at substrate concentrations of 200 microM acetyl-CoA, 150 microM malonyl-CoA, and 200 microM S-adenosylmethionine, around pH 7 and at 35 degrees C. Interestingly the Bmt backbone is released from the enzyme as a coenzyme A thioester, suggesting that subsequent transformation to Bmt takes place upon this activated intermediate.

Dynamic response of immobilized cells to pulse addition of l-valine in cyclosporin A biosynthesis

A feeding strategy for l-valine was tested in the production of cyclosporin A (Cy A), a powerful immunosuppressive secondary metabolite, in celite-immobilized cells of the fungus Tolypocladium inflatum. This system has been previously shown to have promise over conventional submerged systems. Significant increase in Cy A biosynthesis was manifested in the immobilized cells when l-valine was added at 108 h (system C) and at 156 h (system D) during the exponential growth phase. However, no clearly stimulating effect of l-valine on Cy A titre was observed when the amino acid was supplemented at hour 60 (lag phase, system B) or when the valine was present from the start (system A), where system A = 100%, system B = 113%, system C = 253%, system D = 302%. The large contribution to the enhanced production of Cy A in systems C and D may be explained by the preferential channeling of l-valine to growth during the lag phase and to secondary metabolism during the late exponential phase of the immobilized cells.

Mathematical modeling of the production of cyclosporin A by Tolypocladium inflatum: effect of L-valine.

A mathematical model was developed to describe the kinetics of submerged fungal growth, consumption of nutrients, and production of the immunosuppressant drug, cyclosporin A (CyA). Special emphasis was placed on the experimentally observed stimulatory effect of L-valine on CyA production. The proposed model was based on kinetic information and emerging mechanistic data on CyA biosynthesis. It was assumed that L-valine acts as both precursor and inducer of the CyA-synthesizing multienzyme (CyA synthetase), and an unmethylated intermediate of CyA was postulated in the biosynthetic process. The model consisted of two parts: one describing cell growth and substrate consumption and the other addressing the kinetics of internal properties such as endogenous valine, biosynthetic enzyme, CyA intermediate, and CyA. The success of this extensive model was confirmed by its ability to simulate adequately the kinetic profiles of both external and internal variables. For instance, the model correctly predicted the time course of intracellular L-valine accumulation. In addition, both the optimal level and timing of exogenous L-valine addition could be predicted for maximum drug production. This study suggests rational new ways of improving the fungal production process of this important immunosuppressant.

Experimental Evaluation of Ciclosporin A: Cytostatic Effects in a Murine Reticulum Cell Sarcoma (RS-80)

<i>Background:</i> Ciclosporin A (CS-A) is a powerful immunosuppressive agent, isolated from the fungus Tolypocladium inflatum Gams. The value of CS-A in experimental oncology is at present not clear. Studies on xenogeneic tumor transplantation, on the reversal of cytostatic resistance in various tumor models, and on the combination of CS-A with different other cytostatic drugs have been published. <i>Materials and Methods:</i> The 6-day subrenal capsule assay (SRCA) was used. A murine reticulum cell sarcoma was transplanted under the renal capsule of female BlmNMRI mice. Different doses of CS-A (100, 80, 60, 40, and 10 mg/kg) were administered on 6 consecutive days both intraperitoneally and subcutaneously. Tumor size, body weight reduction, and mortality were evaluated.<i>Results:</i> The data demonstrate a significant cytostatic effect of CS-A in the murine reticulum cell sarcoma both after subcutaneous and intraperitoneal administration of CS-A at different doses. A dose-dependent tumor size reduction was found only in the subcutaneously treated animals. The toxicity, measured by body weight reduction, was also significantly higher in the subcutaneously treated animals, but the mortality was higher in the animals treated intraperitoneally with CS-A. Histologic eval-uation showed a reduction of the mitotic activity in all treated versus control groups.<i>Conclusions:</i> The results suggest that CS-A has a cytostatic effect in the murine reticulum cell sarcoma RS-180, transplanted under the subrenal capsule of syngeneic female BlmNMRI mice for 6 days, which is combined with a decrease in tumor size. Histologically, a reduction of mitosis in the groups treated with CS-A was found.

Two new cyclophilins from Fusarium sambucinum and Aspergillus niger: Resistance of cyclophilin/cyclosporin a complexes against proteolysis

Two new peptidyl-prolyl-cis/trans-isomerases were purified to homogeneity from Fusarium sambucinum and Aspergillus niger. They belong to the class of cyclosporin A binding proteins (cyclophilins) and have molecular masses of about 18 kDa. As has been shown for other cyclophilins, the isomerase activity of the enzymes is inhibited by cyclosporin A in the nanomolar range. Furthermore binding of cyclosporin A prevents proteolytic digestion of the cyclophilin/cyclosporin complexes by the endoproteases GluC, LysC and α-chymotrypsin, in contrast to the free cyclophilins, which are readily cleaved by these proteases. We could also observe this protection for cyclophilins from sheep thymus and from the cyclosporin producing fungus Tolypocladium inflatum.

Electrophoretic karyotyping from Tolypocladium inflatum and six related strains allows differentiation of morpologically similar species

The electropheretic karyotype for the imperfect filamentous fungus Tolypocladium inflatum and for six related strains is presented. Pulsed-field gel electrophoresis was used with improved separation conditions to separate DNA from 6.6 Mb to 1.05 Mb in size. Using probes encoding rRNA or the β-tubulin gene from T.iinflatum the corresponding genes were detected on designated chromosomes. In addition, two recombinant lambda clones, carrying T. inflatum chromosomal DNA, were used as chromosome-specific probes. Although all strains investigated are very similar in their morphology, significant chromosome-lengt polymorphisms were detected, allowing easy strain differentiation. The polymorphism was confirmed using an rRNA probe for genomic mapping. All strains contain a homologous “minichromosome” of 1.05 Mb. Finally, the resolution of very large DNA enabled us to separate a 6.6-Mb DNA band that specifically hybridizes with mitochondrial gene probes. The electrophoretic karyotyping presented here may be regarded as a reliable molecular tool to differentiate morphologically very similar filamentous fungi.

Enzymatic biosynthesis of cyclosporin A and analogues

The final assembly of the undecapeptide chain of cyclosporin A and its cycllization is accomplished in Beauveria nivea by cyclosporin synthase. This multienzyme is the largest integrated enzyme structure so far reported. Its size has been estimated at approximately 1 400 kDa by two different methods: 1), by 3% SDS-PAGE using the related multienzymes ACV synthetase and gramicidin S synthetase 2 as references (420 and 556 kDa, respectively); and 2), by CsCl density gradient centrifugation experiments using fluorescence-labeled cyclosporin synthetase. Besides cyclosporin A and a number of cyclosporins known from fermentation studies cyclosporin synthetase is capable of synthesizing some new cyclosporins which are so far unobtainable by fermentation. So, for example the synthesis of [ N- methyl-(+)-2- amino-3- hydroxy-4,4- dimethyloctanoic acid 1] CyA, dihydro-CyA, [ l-norvaline 2,5, N- methyl- lnorvaline 11] CyA, [ l-allo-isoleucine 5, N- methyl- l-allo-isoleucine 11] [ d-2-aminobutyric acid 8] CyA, [β- chloro- dalanine 8] CyA and some related compounds could be established. By using a related but different enzyme from Cylindrotrichum Bonorden, the peptolide [ l-threonine 2, l-leucine 5,10, d-2-hydroxyisovaleric acid 8]CyA could be synthesized in vitro. We were able to synthesize these cyclosporins in sufficient quantities to examine their structure by FAB mass spectroscopy and explore their immunosuppressivity. It was found that all new cyclosporins so far synthesized in the in vitro system are immunosuppressive.

Structure of efrapeptins from the fungus Tolypocladium niveum: peptide inhibitors of mitochondrial ATPase

Efrapeptins, a group of peptide toxins, were isolated from the culture filtrates of the fungus Tolypocladium niveum and complete stereostructures of five efrapeptins C-G were established. The peptides are mitochondrial ATPase inhibitors and have insecticidal properties

FK-506 binding protein from tolypocladium inflatum: Resistance of [formula omitted] complex against proteolysis

A 12-kDa peptidyl-prolyl-cis/trans-isomerase was purified 225-fold to homogeniety from the cyclosporin producing fungus Tolypocladium inflatum. The enzyme is highly sensitive to the immunosuppressant FK-506 but not to cyclosporin and thus belongs to the class of FK-506 binding proteins (FKBP). Interestingly the FKBP FK-506 complex is resistant against proteolytic digestion by the endoproteases GluC and LysC, in contrast to the free FKBP, which is readily cleaved by these proteases. This protection may play a role in the effects of FK-506 in the living cell.

A seventeen kilodaltons peptidyl-prolyl cis-trans isomerase of the cyclosporin-producer Tolypocladium inflatum is sensitive to cyclosporin A.

A peptidyl-prolyl cis-trans isomerase (PPIase) was purified to homogeneity about 24-fold from the cyclosporin-producing fungus Tolypocladium inflatum. The molecular mass of the enzyme was in the range of 17 kdaltons. Remarkably, the enzyme could be inhibited by cyclosporin A in the nanomolar range as has been shown for numerous other cyclophilins from eukaryotic organisms. This indicates, that Tolypocladium inflatum must possess a self protection system in order to survive in the presence of cyclosporin.

Formation of cyclosporins byTolypocladium inflatum

Submerged cultures of the low-production strain of Tolypocladium inflatum DSM 63544 formed a mixture of cyclosporins (CS) consisting of CS-A, CS-B, "CS-3" and "CS-4". Glucose, sucrose and maltose were highly favored for biomass production but provided a different physiological state necessary for CS biosynthesis. Not only the magnitude of CS production but also the proportion of individual components of the CS mixture were affected by the C source. Intensive CS synthesis was in correlation with the formation of CS-3. Lower yields of CS were accompanied by an increased proportion of CS-A in the CS complex. The best specific production of CS was achieved on the glucose medium, the highest yield of CS-A on the maltose medium. There was no remarkable relationship between the biomass formation and the intensity of CS synthesis.

Antifungal and insecticidal properties of the efrapeptins: Metabolites of the fungus Tolypocladium niveum

Efrapeptins, peptides isolated from the fungus Tolypocladium niveum, showed toxic activity, as a crude mixture of five closely related compounds, against twospotted spider mite, Colorado potato beetle,southern armyworm, and tobacco budworm. The insecticidal activity of pure efrapeptins was characterized using two of the compounds against Colorado potado beetle in a foliar spray assay. Efrapeptin F (LC50 = 8.4 ppm) was about twice as potent as efrapeptin D (LC50 = 18.9 ppm). ATPase inhibitory activity of the efrapeptins was demonstrated using mitochondrial preparations from two fungal species, Metarhizium anisopliae and T. niveum, and from an insect, house fly. Against house fly mitochondria, efrapeptin D was the most potent ATPase inhibitor of five efrapeptins tested.

Selective 13C‐labelling of cyclosporin A

Cyclosporin A is biosynthetically labelled with 13C by growing an overproducing strain of Tolypocladium inflatum on minimal media containing either [1-13C]-, [2-13C]-, [3-13C]- or [6-13C]glucose as the only carbon source. NMR analysis of the 13C-labelled peptide showed a labelling pattern in which 13C occurs at specific sites. These can be predicted by consideration of the relevant biosynthetic pathways. Quantitation of the site-specific enrichments revealed that the 13C-label incorporation is efficient and selective. Metabolic fluxes through alternative pathways can also be estimated from these results. Isotopically labelled peptides will be a very useful tool for the study of molecular interactions with their receptors.

The biochemistry of cyclosporin formation: a review

The cyclosporins are a group of over 25 closely related cyclic undercapeptides produced as secondary fungal metabolites by Cylindrocarpum lucidum Booth and Tolypocladium inflatum Gams. Both strains of fungi imperfecti were isolated from soil samples collected in Wisconsin (USA) and Hardanger Vidda (Norway). The isolation, the culture conditions and the taxonomy of these fungi are reported by Dreyfuss et al. 1 Cyclosporin (CS), originally named ‘Cyclosporin A’, is the main component of this family of cyclic peptides each comprising 11 amino acids. The producing strain (NRRL 8044) was initially misidentified as Trichoderma polysporum 2 but is now initially referred to as Tolypocladium niveum. 3 T. niveum has also been classified as Tolypocladium inflatum 4 and as Beauveria nivea. 5 The production of CS by some strains of the phytopathogenic fungus Neocosmospora spp. was described by Dreyfuss. 6 CS was isolated initially for its antifungal activities. Extracts of cultures exhibit in vitro a narrow spectrum of anti-fungal activities but exert only marginal effects in vivo. Borel et al. 7–9 also reported CS activity as an immunosuppressant which acted mainly on Tlymphocytes, showed anti-infammatory effects and was practically devoid of toxicity. It is unique among the presently available immunosuppressive drugs in that it reversibly inhibits only some classes of lymphocytes and does not affect haemopoietic tissues (bone marrow).

Changes in translatable mRNA species associated with nutrient deprivation and protease synthesis in Metarhizium anisopliae

Summary: We have studied the regulation of the extracellular chymoelastase protease (PrI) of the fungal entomopathogen Metarhizium anisopliae. This enzyme is involved in the penetration of insect cuticle by Metarhizium and other entomopathogenic fungi. Changes in mRNA sequences during starvation-induced synthesis of chymoelastase were investigated by comparing poly(A+)RNAs and their complementary DNA in rapidly growing or nutrient-deprived cultures of Metarhizium. Hybrid-selected translation revealed that three novel polypeptides (41, 40·2 and 29·8 kDa) were produced rapidly (<1 h) during nutrient deprivation; the most intensely translated species (41 kDa) was identified as the primary translation product of Pr1 by examination with anti-Pr1-IgG. Measurement of starvation-specific RNA levels by dot-blot hybridization with [32P]cDNA showed that transcripts were not present in spores but were produced when the formation against a hard surface of infection structures was induced by depletion of endogenous nutrient reserves. Starvation-specific mRNA species failed to appear when new RNA synthesis was blocked with actinomycin D, indicating that control is at the level of transcription. Immunoblot analysis with anti-Pr1-IgG showed that Pr1 protein increased in concert with the appearance of Pr1 transcripts and active enzyme. Two very short lived precursors of Pr1, the primary translation product (41 kDa) and an intermediate (40·8 kDa) in addition to mature Pr1 (30 kDa) were detected by immunoprecipitation of [35S]methionine-labelled cell extracts with anti-Pr1-IgG. Pulse-labelling experiments demonstrated that a time of about 7 min was required for [35S]methionine to be processed into extracellular Pr1. These results suggest that regulation of Pr1 gene expression occurs during starvation which, coupled with fast processing, allows very rapid secretion of Pr1. Isolates of four other entomopathogenic fungi (Verticillium lecanii, Beauveria bassiana, Tolypocladium niveum and Paecilomyces farinosus) produced Pr1-type enzymes during nutrient deprivation, suggesting our results may have widespread application in understanding entomopathogenicity.

Comparative studies of physiological and environmental effects on the production of cyclosporin A in suspended and immobilized cells of Tolypocladium inflatum

Process improvement of the production of cyclosporin A (Cy A), a powerful immunosuppressive fungal metabolite, has been undertaken by analyzing suspended and immobilized cell cultures in parallel. Conidiospores of the producer microorganism, Tolypocladium inflatum, were entrapped into porous celite particles. Easier germination of the entrapped spores and more active growth of the immobilized cells were manifested when compared with free cell cultures initiated with spores or with mycelial inocula. Significant differences in precursor flow between the immobilized and free cell systems were evident when the effects of L-valine (a constituent amino acid of the Cy A molecule) on Cy A biosynthesis were compared in the two systems. For the freely suspended cells, L-valine supplemented early in the fermentation served as a possible precursor or stimulator of Cy A biosynthesis. A significant increase in specific production and Cy A yield on carbon source was observed in this system relative to suspended cultures supplemented with L-valine during or after exponential growth. In contrast to the free cell cultures, the addition of L-valine during the initial stage of immobilized cell growth had a negative effect on Cy A production but resulted in somewhat increased cell growth. This suggests an incompatibility between primary and secondary metabolic networks involved in Cy A biosynthesis in the immobilized state upon external addition of the amino acid.

Dynamics of L-valine in relation to the production of cyclosporin A by Tolypocladium inflatum.

We report the kinetics of endogenous L-valine in the fungus Tolypocladium inflatum, in an effort to understand the enhancing effect of externally supplemented L-valine on the production of the immunosuppressant cyclosporin A (CyA) in chemically defined medium. In a batch laboratory stirred reactor cultivation, the concentration of intracellular L-valine increased by up to four times between the end of the exponential phase and the beginning of the stationary phase when the medium was supplemented externally with 4 g/l L-valine. The final CyA titre under these conditions was 710 mg/l compared to only 130 mg/l attained without L-valine supplementation. In contrast to substantial growth-associated production of CyA in unsupplemented culture, the formation of the immunosuppressant was prolonged during the stationary phase in L-valine-supplemented medium. As a result, the conversion yield of CyA on L-valine remained constant during the stationary phase at 0.27 g CyA/g L-valine.