Production of extracellular enzymes and cyclosporin byTolypocladium inflatum and morphologically related fungi

Summary Tolypocladium inflatum, the producer of cyclosporins, and morphologically related fungi from the generaTolypocladium, Beauveria, Fusarium andNeocosmospora produced different levels of cyclosporin A and of extracellular lipase and chitinase. However, entomopathogenically important protease activity was not detected.     

Influence of amino acids on the biosynthesis of cyclosporin A by Tolypocladium inflatum

 An indigenously isolated strain of Tolypocladium inflatum, when grown as a suspension culture in semi-synthetic and synthetic media, produced cyclosporin A. Biosynthesis of this well-known immunosuppressive agent was found to be influenced heavily by the external addition of the amino acid constituents of the molecule. In synthetic media, L-leucine and L-valine were found to act as strong inducers of drug production. L-Valine increased the specific production of cyclosporin A by 75% in semi-synthetic medium and by ten times in synthetic medium compared to an unsupplemented control culture. D-Valine had no stimulating effect on the production. The presence of amino acids in the exponential growth phase ensured optimal production, as was indicated in the experiment in which L-valine was added at different times; 4 g/l was the optimum concentration of exogenous L-valine. On the other hand, exogenous sarcosine and L-methionine tended to diminish drug production. Received: 23 October 1995/Received revision: 23 January 1996/Accepted: 29 January 1996     

Formation of cyclosporins byTolypocladium inflatum

Submerged cultures of the low-production strain ofTolypocladium 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.     

Growth and cyclosporin a production by an indigenously isolated strain ofTolypocladium inflatum

A fungal culture isolated from a local soil sample which showed antifungal activity and produced cyclosporin A, was identified asTolypocladium inflatum. The culture grew best in a medium containing 1% maltose (pH 5–6) when inoculated with a one-day-old inoculum at 2% (V/V) concentration. Under batch fermentation conditions, growth and cyclosporin A production were better in complex media (24.6 g biomass and 205 mg cyclosporin A per liter) in comparison with synthetic media (6.84 g biomass and 35 mg cyclosporin A per liter). While addition of peptone increased culture growth (high biomass yield), supplementation with casein acid hydrolyzate favored cyclosporin A production.     

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     

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.     

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 glutathione production by Saccharomyces cerevisiae

Summary The constituent amino acids of the glutathione (GSH) tripeptide chain, glutamate, cysteine and glycine, were investigated for positive effects on GSH production in shake-flask cultures of Saccharomyces cerevisiae with glucose as the carbon source. Cysteine was confirmed as the key amino acid for increasing the specific GSH production rate, g, but showed some growth inhibition, especially in the second growth phase (ethanol-assimilation phase). An intracellular cysteine delivery agent, thiazolidine, showed a similar pattern of increased GSH production and growth inhibition, but to a slightly lesser degree, compared with free cysteine. The initial cysteine concentration affected both the specific growth rate, µ, and g, up to about 5 mm for µ and about 2–3 mm for g. Results of the [³⁵S]cysteine-labelling experiments suggest a complicated role of cysteine in increasing GSH production and further investigation may be necessary. Offprint requests to: S. Shioya     

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.     

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.     

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.     

Effect of various amino acids and ammonium salts in a synthetic culture media on the cholera enterotoxin production

The influence of amino acids and ammonium salts on the production of cholera enterotoxin (CT) by 3 Vibrio cholerae strains of different biovars and serogroups was evaluated. As revealed in this study, toxin formation in each of the strains was quantitatively and qualitatively determined by their individual sets of amino acids. The amino acid compositions ensuring the maximum production of CT by the V. cholerae strains under study were formed. The use of ammonium salts as the only source of nitrogen in the composition of a synthetic nutrient medium for the accumulation of CT was shown to be inexpedient.     

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.     

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.     

Effect of amino acids and vitamins on laccase production by the bird's nest fungus Cyathus bulleri

Various amino acids, their analogues and vitamins have shown stimulatory as well as inhibitory effects on laccase production by Cyathus bulleri. DL-methionine, DL-tryptophan, glycine and DL-valine stimulated laccase production, while L-cysteine monohydrochloride completely inhibited the enzyme production. Among vitamins tested biotin, riboflavin and pyridoxine hydrochloride were found to induce laccase production.