Structure and localization of cyclosporin synthetase, the key enzyme of cyclosporin biosynthesis in Tolypocladium inflatum

The electron microscopic image of native cyclosporin synthetase molecules showed large globular complexes of 25 nm in diameter, built up by smaller interconnected units. Compartmentation of cyclosporin synthetase and the functionally interconnected D-alanine racemase was revealed after sucrose density gradient centrifugation of subcellular fractions and immunoelectron microscopy. A considerable proportion of cyclosporin synthetase and D-alanine racemase was detected at the vacuolar membrane. The product cyclosporin was localized in the fungal vacuole.     

Pigmented variants of Tolypocladium inflatum in relation to cyclosporin A production

Summary We have isolated four distinct colony types of the fungus Tolypocladium inflatum, the producer of the immunosuppresive agent cyclosporin A: morphologically normal white, red, and orange colonies and morphologically diverse tiny brown colonies. In liquid cultures, white normal and brown colonies developed into yellow broths. The broth of the brown colony had a low final pH and low cyclosporin production, whereas orange and red colonies had dark brown and even black broths with higher final pH and high cyclosporin production. The specific production of cyclosporin A by the red colony was three times that of the white normal colonies. Offprint requests to: S. N. Agathos     

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

Summary 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/1 l-valine. The final CyA titre under these conditions was 710 mg/1 compared to only 130 mg/1 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.     

Effect of amino acids on the production of cyclosporin a byTolypocladium inflatum

Summary Cyclosporin A was produced in semi-synthetic and synthetic media byTolypocladium inflatum in suspension culture. L-valine increased the specific production of cyclosporin A by 62% in semi-synthetic media and by 4 times in synthetic media, compared to an unsupplemented control culture. D-valine had no stimulatory effect on the production. L-leucine was also a strong enhancer of drug production in synthetic media. Experiments with different times of addition of L-valine indicated that the amino acid may need to be present in exponential growth phase for optimal production. 4g/l was the optimum concentration of exogenous L-valine. In contrast, exogenous L-methionine and sarcosine tended to diminish the production of cyclosporin A.     

Application of factorial experimental designs for optimization of cyclosporin. A production by Tolypocladium inflatum in submerged culture

A sequential optimization strategy based on statistical experimental designs was employed to enhance the production of cyclosporin A (CyA) by Tolypocladium inflatum DSMZ 915 in a submerged culture. A 2-level Plackett-Burman design was used to screen the bioprocess parameters significantly influencing CyA production. Among the 11 variables tested, sucrose, ammonium sulfate, and soluble starch were selected, owing to their significant positive effect on CyA production. A response surface methodology (RSM) involving a 3-level Box-Behnken design was adopted to acquire the best process conditions. Thus, a polynomial model was created to correlate the relationship between the three variables and the CyA yield, and the optimal combination of the major media constituents for cyclosporin A production, evaluated using the nonlinear optimization algorithm of EXCEL-Solver, was as follows (g/l): sucrose, 20; starch, 20; and ammonium sulfate, 10. The predicted optimum CyA yield was 113 mg/l, which was 2-fold the amount obtained with the basal medium. Experimental verification of the predicted model resulted in a CyA yield of 110 mg/l, representing 97% of the theoretically calculated yield.     

Enhancement of cyclosporin production in a Tolypocladium inflatum strain after epichlorohydrin treatment.

Following treatment of conidia of the cyclosporin producer fungus, Tolypocladium inflatum, with 0.15 M epichlorohydrin, strain M6 was isolated. The new strain exhibited a similar growth rate to the parent organism but more extensive conidiation and several-fold higher overall cyclosporin production. Strain M6 reached titres of 318 mg l-1 cyclosporin A in agar cultures, whereas in liquid medium it produced 140 mg l-1 cyclosporin A and 68 mg l-1 cyclosporin C. It also maintained a steady volumetric productivity of 0.48 mg l-1 h-1 cyclosporin A over 2 weeks of submerged cultivation in maltose-based semisynthetic medium. The new strain holds potential for improved cyclosporin production due to the superior titres and demonstrated capacity to sustain elevated production of cyclosporin for periods greater than the wild type.     

Biosynthesis of cyclosporin A: partial purification and properties of a multifunctional enzyme from Tolypocladium inflatum

An enzyme fraction most probably involved in the biosynthesis of cyclosporin A was purified 540-fold from Tolypocladium inflatum. The enzyme was capable of forming covalent enzyme-substrate complexes and catalyzed the ATP-pyrophosphate exchange reactions dependent on the unmethylated constituent amino acids of cyclosporin A. Evidence was obtained that covalent binding of substrate amino acids occurred via thioester linkage. Furthermore, the N-methylation of thio-esterified valine, leucine, and glycine residues with S-adenosyl-L-methionine was demonstrated. De novo synthesis of cyclosporin A was not observed but the formation of the diketopiperazine cyclo-(D-Ala-MeLeu) from D-alanine and L-leucine under the consumption of ATP and S-adenosyl-L-methionine. This cyclodipeptide represents a partial sequence of cyclosporin A. Molecular mass determinations revealed the enzyme activity to be lying in the range of about 700 kDa.     

Kinetics of cell growth and cyclosporin A production by Tolypocladium inflatum when scaling up from shake flask to bioreactor

The kinetics of cell growth and Cyclosporin A (Cyc A) production by Tolypocladium inflatum were studied in shake flasks and bioreactors under controlled and uncontrolled pH conditions. In the case of the shake flask, the production time was extended to 226 h and the maximal antibiotic concentration was 76 mg/l. When scaling up the cultivation process to a bioreactor level, the production time was reduced to only 70 h with a significant increase in both the cell growth and the antibiotic production. The maximal dry cell weights in the case of the controlled pH and uncontrolled pH cultures in the bioreactor were 22.4 g/l and 14.2 g/l, respectively. The corresponding maximal dry cell weight values did not exceed 7.25 g/l with the shake flask cultures. The maximal values for Cyc A production were 144.72 and 131.4 mg/l for the controlled and uncontrolled pH cultures, respectively. It is also worth noting that a significant reduction was observed in both the dry cell mass and the antibiotic concentration after the Cyc A production phase, whereas the highest rate of antibiotic degradation was observed in the stirred tank bioreactor with an uncontrolled pH. Morphological characterization of the micromorphological cell growth (mycelial/pellet forms) was also performed during cultivation in the bioreactor.     

From Norway to Novartis: cyclosporin from Tolypocladium inflatum in an open access bioprospecting regime

The Convention on Biological Diversity (CBD) introduces a new regime of source countries' national sovereignty over genetic resources, in which benefit sharing is a central factor. This article shows how Tolypocladium inflatum was collected in Norway in 1969 within an open access regime implying that there is no benefit sharing with the source country from Novartis' present sales of the derived medicines based on cyclosporin. We estimate source country's loss of benefits in comparison with present norms and expectations concerning bioprospecting. Two percent annual royalties would have been a reasonable claim in this case, and in 1997 this amounted to US$ 24.3 million. Such benefits could, for instance, have been targeted to conservation, scientific capacity building and health care. The study provides an indication of possible gains for source countries – countries with developed as well as developing economies – in a case of the finding of a blockbuster drug. Institutional prerequisites for benefit sharing are discussed, and the emphasis, which often is placed on the role of patents as the cause of lack of source country benefits, is in this case found to be misleading.     

Cyclosporin-A production by Tolypocladium inflatum using solid state fermentation

Tolypocladium inflatum strains are known to produce Cyclosporin-A under submerged culture conditions. In the present study solid state fermentation was used to produce Cyclosporin-A. Tolypocladium inflatum strains when grown on moist wheat bran produced 310–459 mg of Cyclosporin-A kg of bran⁻¹. Tolypocladium inflatum ATCC 34921 which produced 459 mg of Cyclosporin-A kg of bran⁻¹ was improved to produce 1031±27 mg of Cyclosporin-A kg⁻¹ of bran, by subjecting the spores to different mutagenic treatments. The mutated strain, designated Tolypocladium inflatum DRCC 106, produced 4843 mg kg⁻¹ of bran under optimum fermentation conditions in 10 days when grown on wheat bran medium containing millet flour 20%, jowar flour 10%, zinc sulphate 0·15%, ferric chloride 0·25% and cobaltous chloride 0·05%. An inoculum of 60% initial moisture content 70%, initial bran pH 2·0 and incubation temperature 25°C were found to be optimal. Cyclosporin-A thus obtained was purified by solvent extraction, followed by column chromatography. The isolated product complies with the United States Pharmacopoeia specifications.     

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.     

Effect of amino acids on thaxtomin A biosynthesis by Streptomyces scabies

The regulatory effect of amino acids on the production of thaxtomin A, a phytotoxin produced by Streptomyces scabies, was investigated. Tryptophan had an important inhibitory effect on the toxin biosynthesis in all five strains of S. scabies tested. Two other aromatic amino acids (tyrosine and phenylalanine) also inhibited thaxtomin A biosynthesis, while aliphatic amino acids did not cause an important decline in thaxtomin A production. Methylation of tryptophan prevented or reduced the inhibitory effect on thaxtomin A biosynthesis. In spite of the inhibitory action of tryptophan and phenylalanine on thaxtomin A production, incorporation of these radiolabeled molecules into thaxtomin A confirmed that they are metabolic precursors for the biosynthesis of the phytotoxin.     

Tagging of a nitrogen pathway-specific regulator gene in Tolypocladium inflatum by the transposon Restless

Restless is an endogenous hAT transposon found in the cyclosporin-producing fungus Tolypocladium inflatum. This element is present in about 15 copies in a particular strain (ATCC34921) which was used for successful gene tagging. We have isolated a T. inflatum mutant with a defect in nitrogen metabolism. This mutant carries a copy of the Restless element in a gene encoding a C6 zinc-finger protein. The deduced amino acid sequence of the gene product shows a significant similarity to the NIT4 protein of Neurospora crassa, which is a regulator of nitrogen metabolism. The wild-type T. inflatum gene was shown to complement a nit-4 mutant of N. crassa. From these data, we conclude that the T. inflatum gene also encodes a regulator of nitrogen metabolism, which was named tnir1 (Tolypocladium nitrogen regulator 1). To the best of our knowledge, this is the first fungal gene to be identified by transposon-directed gene tagging. A general method for gene tagging using an endogenous fungal transposon is presented. Received: 20 October 1999 / Accepted: 15 December 1999     

Lovastatin effect on ergosterol production and growth of Tolypocladium inflatum 106]

The effect of lovastatin on Tolypocladium inflatum 106 was studied. The strain was shown to be highly sensitive to lovastatin when its MIC was determined by the agar diffusion method and under submerged conditions that was considered possible to use the strain as a test culture in screening of new natural compounds with hypolipidemic action and to study its specificity. It was demonstrated that the effect of lovastatin on ergosterol synthesis in T. inflatum 106 was of specific dose-dependent polymodal nature.     

Effect of temperature on the extraction kinetics and diffusivity of Cyclosporin A in the fungus Tolypocladium inflatum

The influence of temperature on the extraction kinetics of Cyclosporin A (CyA) from the mycelia of Tolypocladium inflatum was examined in this study. The extraction of CyA from mycelia was performed in a 2-L stirred, baffled vessel using 30% v/v aqueous methanol. The temperature range used was from 5 to 45 degrees C. A linear relationship was found between the extraction yield of CyA and temperature. As the temperature increased, the yield of CyA increased with a maximum CyA yield of 18.3% obtained at 45 degrees C, which is 21.3% higher than the yield at 25 degrees C. The activation energy for the extraction of CyA from T. inflatum was found to be 36.7 kJ/mol, which indicates that the extraction of CyA from T. inflatum is controlled by both solubilization of CyA and diffusion of CyA through the solid phase of mycelia. The overall mass transfer coefficient, k(L)a(S), was found to increase from 1.02 x 10(-3) to 1.34 x 10(-2) s(-1) as the temperature increased from 5 to 45 degrees C. The effective diffusivity of CyA in the solid matrix of mycelia was found to increase from 1.05 x 10(-15) to 1.43 x 10(-14) m(2)/s as the temperature increased from 5 to 45 degrees C. A mathematical diffusion model was developed and was used to fit the experimental kinetic data of CyA extraction and determination of CyA effective diffusivities at different temperatures. This is the first time CyA diffusivities as a function of extraction temperature are reported in the literature.     

Effect of solvent concentration on the extraction kinetics and diffusivity of Cyclosporin A in the fungus Tolypocladium inflatum

The kinetics of solid-liquid extraction and extraction yields of the immunosuppressant drug Cyclosporin A (CyA) from the mycelia of Tolypocladium inflatum were examined in this study. A 2 L stirred, baffled vessel was used to extract CyA from wet mycelia mass. Three different organic solvents were used, namely, methanol, acetone, and isopropanol at different concentrations in aqueous mixtures at room temperature. It was found that the best solvent was acetone at 50% v/v concentration achieving 100% extraction of CyA from the mycelia of T. inflatum. Although acetone proved to be the better solvent for CyA extraction, further studies were performed using methanol. A linear relationship was found between extraction yield of CyA and methanol concentration with 100% CyA extraction at 90% v/v methanol. The partition coefficients of CyA between the solid mycelia phase and the aqueous solvent phase were found to decrease exponentially with increasing methanol concentration. A liquid extraction model was developed based on the diffusion equation to correlate the kinetic data of CyA extraction from the solid mycelia of T. inflatum. Non-linear regression analysis of experimental data was used with the diffusion equation in order to calculate the effective diffusivities of CyA in the mycelia of T. inflatum. For all three organic solvents used, the effective diffusivities of CyA were found to be between 4.41 x 10(-15) and 6.18 x 10(-14) m(2)/s. This is the first time CyA effective diffusivities in T. inflatum are reported in the literature.     

Effect of amino acids on rapamycin biosynthesis by Streptomyces hygroscopicus

In a chemically defined medium containing aspartate, arginine and histidine to support good growth, addition of L-lysine stimulated rapamyycin production by 150%. This was probably due to its conversion to pipecolic acid, a rapamycin precursor. Phenylalanine and methionine interfered in rapamycin production by unknown mechanisms.     

Effect of amino acids on collagen biosynthesis

Summary The effect of various amino acids on collagen biosynthesis was studied in organ cultures of chicken embryo tibiae. Competitive interrelationship between selected amino acids influences independently the uptake of proline and lysine, precursors of hydroxyproline and hydroxylysine, respectively, which are the two amino acids characteristics of collagen. On the basis of these influences, the possibility of biosynthesis of anomalous collagens is stressed. Parallel studies of biosynthesis of hydroxyproline and hydroxylysine are necessary.     

Rheology and Hydrodynamic Properties of Tolypocladium inflatum Fermentation Broth and Its Simulation

A physico-chemical, two phase simulated pseudoplastic fermentation (SPF) broth was investigated in which Solka Floc cellulose fibre was used to simulate the filamentous biomass, and a mixture of 0.1% (w/v) carboxymethyl cellulose (CMC) and 0.15 M aqueous sodium chloride was used to simulate the liquid fraction of the fermentation broth. An investigation of the rheological behaviour and hydrodynamic properties of the SPF broth was carried out, and compared to both a fungal Tolypocladium inflatum fermentation broth and a CMC solution in a 50 L stirred tank bioreactor equipped with conventional Rushton turbines. The experimental data confirmed the ability of the two phase SPF broth to mimic both the T. inflatum broth bulk rheology as well as the mixing and mass transfer behaviour. In contrast, using a homogeneous CMC solution with a similar bulk rheology to simulate the fermentation resulted in a significant underestimation of the mass transfer and mixing times. The presence of the solid phase and its microstructure in the SPF broth appear to play a significant role in gas holdup and bubble size, thus leading to the different behaviours. The SPF broth seems to be a more accurate simulation fluid that can be used to predict the bioreactor mixing and mass transfer performance in filamentous fermentations, in comparison with CMC solutions used in some previous studies.