Development of a cell-loaded biosupport separator for continuous immobilized-cell perfusion culture.

An efficient cell-loaded biosupport separator of the decantor type was developed and applied for a continuous perfusion culture to produce cyclosporin A (CyA), in which fungal cells were immobilized on Celite beads. In the preliminary experiments employing highly viscous polymer (carboxymethyl cellulose) solutions, the decantor showed good separation performances at high solution viscosites and dilution rates. Two concentric cylindrical tubes installed inside the decantor turned out to play key roles in the efficient separation of the immobilized cells. By installing the decantor on an immobilized-cell perfusion bioprocess system, a stable continuous operation was possible even at a high dilution rate for an extended period, leading to high productivities of free cells and CyA. Almost no immobilized biomass existed in the effluent stream from the bioreactor, demonstrating the effectiveness of the decantor system. It is noteworthy that we could obtain these results despite unfavorable fermentation conditions, i.e., reduced apparent density of cell-loaded beads and increased drag force on the bead particles caused by overgrowth of cells on the bead surface, tubulence caused by large air bubbles, and the existence of a high density of suspended fungal cells (10 g/L) in the fermentation broth.

[Effect of cyclosporin A and amphotericin B on protoplasts of Tolypocladium inflatum]. / Vliianie tsiklosporina A i amfoteritsina B na protoplasty Tolypocladium inflatum.

When added to the suspension of washed cells of Tolypocladium inflatum (cyclosporine-producing organism) in a concentration up to 100 micrograms/ml, cyclosporine A had no effect on permeability of the cell covers as judged by the quantity of the metabolites with the absorption maxima at 260 nm that leaked to the medium. When cyclosporine was added to the suspension of the T-inflatum cell protoplasts there was a significant increase in the metabolites leaking. Impairment of the permeability of the protoplast membranes was more marked in low potent strains. Impairment of the protoplast membrane permeability by amphotericin B was equal in both the high and the low potent strains and in this respect it was more active than cyclosporine.

Development of sporulation/immobilization method and its application for the continuous production of cyclosporin A by Tolypocladium inflatum.

An efficient sporulation/immobilization procedure for immobilized fungal cell culture was developed by modifying an existing immobilized technique to shorten the time and number of steps for sporulation. This method was applied to an immobilized-cell perfusion bioprocess (IPB) for continuous production of CyA, an intracellular secondary metabolite produced by a filamentous fungus, Tolypocladium inflatum. In the IPB, the fungal cells were immobilized in the pores of celite beads (100-500 microm) and a top-driven stirred tank fermentor was used for the culture. The IPB showed good process benefits as demonstrated by the high density of immobilized cells continuously producing CyA-containing free cells. The productivity of cyA-containing free cells in the effluent was very high, ca. 1.0g/(L/h) at a dilution rate of 0.1 h-1, due to the high density of immobilized cells in the fermentor. The CyA productivity was 4.0-6.0 mg/(L/h) which was about 6-10-fold higher than that of batch suspended cell culture. Such an efficient IPB was possible since a decantor was developed in this study, which could effectively separate cell-immobilized beads from the effluent although bead loss slightly increased as the cell loading increased in the latter part of culture. Furthermore, long-term operation of IPB was carried out successfully by employing an in-situ immobilization strategy. It was found that a large number of spores in the fermentation broth in the reactor were entrapped in-situ into the newly supplemented celite beads and then germinated, thus forming new immobilized cells.

Reversible denaturation of cyclosporin synthetase by urea.

The reversible denaturation of the multifunctional polypeptide, cyclosporin synthetase, by urea was analyzed. It is possible to discriminate between at least two stages of enzyme denaturation. While at low urea concentration (up to 0.8M) cyclosporin A formation is inhibited, synthesis of the diketopiperazine cyclo-(D-alanyl-N-methylleucyl), a molecule representing a partial sequence of cyclosporin A is still detectable. At higher concentrations of urea the enzyme preparation is totally inactive. This inactivation is a consequence of conformational change(s) of cyclosporin synthetase as shown by fluorescence emission spectra of native and denatured enzyme. These data imply a consecutive folding/defolding mechanism for the different domains forming the multifuntional polypeptide.

Growth and cyclosporin A production by an indigenously isolated strain of Tolypocladium inflatum.

A fungal culture isolated from a local soil sample which showed antifungal activity and produced cyclosporin A, was identified as Tolypocladium 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.

Cloning and sequencing of a cyclophilin gene from the cyclosporin producer Tolypocladium niveum.

The gene of a 19 kDa cyclophilin of Tolypocladium niveum was isolated and sequenced. The open reading frame of 956 bp is interrupted by four introns of 76, 221, 58, and 61 bp size. 101 nucleotides upstream of the start codon ATG we found a putative promoter region containing a TATA and a CAAT box. The predicted amino acid sequence shows high identity to other cyclophilins, especially to the cyclophilin A family members of eucaryotic and procaryotic origin. Similar to the case of N. crassa we found a segment of 13 additional amino acid residues near the N-terminus which is not present in the other cyclophilins.

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.

[Effect of H-ATPase stimulators on cyclosporin biosynthesis]. / Vliianie stimuliatorov H-ATFazy na biosintez tsiklosporina.

An analogy was observed between the mechanisms of action of phytohormones on plant cells and cells of the fungus producing cyclosporine. Fusicoccin and cytokinin were shown to have a high stimulating action on the biosynthesis of cyclosporine. The stimulating concentrations of the phytohormones and the time of their maximum effect were determined. The electron microscopic studies demonstrated that an increase in the level of the cyclosporine synthesis correlated with a significant increase in the number of the cells in the state of the coagulation necrosis.

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.

Mechanism of cyclosporin A biosynthesis. Evidence for synthesis via a single linear undecapeptide precursor.

Cyclosporin A is synthesized by cyclosporin synthetase, a multienzyme polypeptide. This enzyme catalyzes at least 40 reaction steps in an assembly belt-like mechanism. It activates all constituent amino acids of cyclosporin A to thioesters via amino acyladenylates and carries out specific N-methylation reactions. During elongation, the activated amino acids are linked by peptide bonds leading to enzyme-bound nascent peptide chains. Some of the linear peptides of the growing cyclosporin A chain were isolated and their N-terminal amino acid was determined. D-Alanine at position 8 of the cyclosporin A molecule was found to be a starting amino acid in the biosynthetic process of cyclosporin A formation. Four intermediate peptides of the growing peptide chain of cyclosporin A could be isolated and identified. All of them represent partial sequences of cyclosporin A starting with D-alanine. That these intermediate peptides were bound by thioester linkage to cyclosporin synthetase could be demonstrated by liberation of the peptides with performic acid. The peptides strongly suggest the stepwise synthesis of a single linear peptide precursor of cyclosporin A.

FR901459, a novel immunosuppressant isolated from Stachybotrys chartarum No. 19392. Taxonomy of the producing organism, fermentation, isolation, physico-chemical properties and biological activities.

FR901459, a novel immunosuppressant, has been isolated from the fermentation broth of Stachybotrys chartarum No. 19392. The molecular formula of FR901459 was determined as C62H111N11O13. FR901459 was found to be a member of the cyclosporin family. However, it is structurally distinct from any other cyclosporins discovered so far, in that Leu is present at position 5 instead of Val. FR901459 was capable of prolonging the survival time of skin allografts in rats with one third the potency of cyclosporin A.

Substrate specificities of cyclosporin synthetase and peptolide SDZ 214-103 synthetase. Comparison of the substrate specificities of the related multifunctional polypeptides.

The recently discovered multifunctional polypeptide cyclosporin synthetase is capable of synthesizing the cyclic undecapeptide cyclosporin A in a batch reaction. Substrates are the unmethylated constitutive amino acids of cyclosporin A. Exchange of one or more of these by various amino acids gives a picture of the substrate specificity of the enzyme in vitro, which is different from the known picture obtained by in vivo studies. The uncommon amino acid butenylmethylthreonine in position 1 of the cyclosporin ring can be exchanged by an unexpected large spectrum of different amino acids, showing a great flexibility of this site. Position 2, on the other hand, which shows the greatest variability in vivo, has an only slightly lower specificity in vitro. Position 3 has a very high degree of specificity; positions 4, 6, 7, 9, and 10 have marginally less. The variability of positions 5 and 11 is moderate, whereas position 8 shows only low substrate specificity in vitro. In general, most sites of SDZ 214-103 synthetase appear to be more specific than those of cyclosporin synthetase. Site 11 has nearly identical substrate specificity compared with that of cyclosporin synthetase. The D-2-hydroxy acid position (position 8) can be occupied by a large spectrum of substrates varying from D-lactic acid to D-2-hydroxyisocaproic acid. Within the limits of the present data, the addition of further functional groups to the D-2-hydroxy acid moiety are apparently not tolerated by the enzyme.

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.

Enzymatic biosynthesis of cyclosporin A and analogues.

The final assembly of the undecapeptide chain of cyclosporin A and its cyclization is accomplished in Beauveria nivea by cyclosporin synthetase. 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 acid1]CyA, dihydro-CyA, [L-norvaline2,5, N-methyl-L-norvaline11]CyA, [L-allo-isoleucine5, N-methyl-L-allo-isoleucine11]CyA, [D-2-aminobutyric acid8]CyA, [beta-chloro-D-alanine8]CyA and some related compounds could be established. By using a related but different enzyme from Cylindrotrichum Bonorden, the peptolide [L-threonine2, L-leucine5,10, D-2-hydroxyisovaleric acid8]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.

[Characteristics of obtaining protoplasts from 2 strains of Tolypocladium inflatum subsp. Blastosporum]. / Osobennosti obrazovaniia protoplastov u dvukh shtammov Tolypocladium inflatum subsp. Blastosporum.

The optimal conditions for preparing protoplasts with high yields by using the cells of two (low and high potent) isogenic cyclosporine-producing Tolypocladium strains were developed. A specific medium containing 0.5 per cent yeast autolysate (by dry weight) and 3 per cent glucose was used. When grown on this medium the cells of the highly potent strain 847 acquired a yeast-like shape. High yields of protoplasts prepared from the low potent strain 43 mycelium were obtained via prior incubation with 0.01 M dithiothreitol followed by treatment with a complex of enzymes from Helix pomatia for 1.5 to 2 hours was used. For preparation of the protoplasts with employing the highly potent strain 847 cells the prior incubation with dithiothreitol was not required, but it was necessary to employ a mixture of the enzyme complex (Helix pomatia), drizilase (Irpex lacteus) and chitinase (Streptomyces griseus) for 18 hours. The electron microscopic data on the two isogenic strains and their protoplasts are presented. The protoplasts proved to be a suitable initial material for investigating bioenergetic processes at the subcellular level and further genetic improvement of the strains.

[Activity of glycolysis enzymes in cyclosporine-producing Tolypocladium sp]. / Aktivnost' fermentov glikoliza u Tolycocladium sp.-produtsenta tsiklosporina.

Enzymes of various glycolysis stages, i.e. hexokinase, phosphofructokinase, pyruvate kinase and lactate dehydrogenase, were detected in cyclosporine-producing organisms belonging to Tolypocladium. The initial activity of the enzymes in the highly active strain was much higher than that in the starting low active strain. During the fermentation the activity of the glycolysis enzymes per 1 mg of mycelium protein in the both strains increased. This was accompanied by a decrease in respiration activity. Therefore, there was a direct correlation between cyclosporine biosynthesis and glycolytic activity of the mycelium in Tolypocladium sp.

Cell-free biosynthesis of cyclosporin A and analogues.

The final assembly of the 11-peptide chain of cyclosporins and its cyclization is accomplished in the producer Beauveria nivea by cyclosporine synthetase. This multienzyme represents the largest integrated enzyme structure reported so far. Its size has been estimated to approximately 1,500 kDa by SDS-PAGE. Some enzyme bound linear peptides could be isolated and identified. All of them represent partial sequences of cyclosporin A starting with D-alanine. These peptides were bound by thioester linkage to the enzyme. This could be demonstrated by liberation of the peptides with performic acid and by inhibition of in vitro cyclosporin A synthesis with thiol blocking agents. Cyclosporin synthetase is capable to synthesize besides a lot of cyclosporins known from fermentation studies some new cyclosporins so far not obtainable by fermentation. So, for example the synthesis of [N-methyl-(+)-2-amino-3-hydroxy-4,4-dimethyloctanoic acid1]CyA, Dihydro-CyA, [L-norvaline2,5, N-methyl-L-norvaline11]CyA, [L-allo-isoleucine5, N-methyl-L-allo-isoleucine11]CyA, [D-2-aminobutyric acid8]CyA, [beta-chloro-D-alanine8]CyA and some related compounds could be established. We were able to synthesize these cyclosporins in sufficient quantities to ensure their structure by fast atom bomdardment mass spectroscopy and to examine their immunosuppressitivity. All new cyclosporins synthesized in the in vitro system so far are immunosuppressive.