production of cyclosporins by tolypocladium niveum strains

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 1990, p. 121-127 Vol. 34, No. 10066-4804/90/010121-07$02.00/0Copyright © 1990, American Society for Microbiology

Production of Cyclosporins by Tolypocladium niveum StrainsCHARLES E. ISAAC,t ALAN JONES,t AND MICHAEL A. PICKARD*

Department of Microbiology, University of Alberta, Edmonton, Alberta, Canada T6G 2E9

Received 1 May 1989/Accepted 2 October 1989

Nine strains of Tolypocladium niveum (=inflatum) were compared for their production of cyclosporins. Twoof the strains, which were originally from the parental NRRL 8044 strain, were among the lower producers,while seventeen Tolypocladium strains belonging to seven other species produced no detectable cyclosporins.Variable cyclosporin production was observed initially. Once extraction and quantitation methods had beenestablished, spore inoculum density and cultural morphology and carbon and nitrogen sources were found tobe among the variables affecting cyclosporin production. Cyclosporin A was identified by cochromatographyby using high-performance liquid chromatography, and cyclosporins A, B, and C were identified by gaschromatography-mass spectroscopy; all three compounds exhibited biological activity. They were routinelyproduced as a mixture in the ratio 7:1:2 in T. niveum UAMH 2472, which was selected on the basis ofsingle-spore isolate total cyclosporin production and was used for most studies. This strain routinely producedtotal cyclosporin levels of 150 to 200 mg. liter-' after 12 days of growth on a 2% sorbose-1% vitamin assayCasamino Acids medium.

Cyclosporins are a family of cyclic undecapeptides, con-taining some unusual amino acids, which can be produced ina fungal fermentation process. The producing strain (NRRL8044) was initially misidentified as Trichoderma polysporum(19) but is now referable to Tolypocladium niveum (6). T.niveum (4) has also been classified as Tolypocladium in-flatum (12) and as Beauveria nivea (22). The classification asT. niveum (20) is used in this study. Cyclosporins are onlymildly effective as antibiotics, but the clinical use of cyclo-sporin A as an immunosuppressant to prevent rejection oftransplanted organs has become widespread. There is alsopotential clinical use in the treatment of various autoimmunediseases (5). The mode of action of cyclosporin is beingstudied, and it seems likely that the enzyme peptidyl-prolylcis-trans isomerase, which is involved in the folding ofproteins, is involved. Recent data (10, 21) indicate that thisisomerase is identical to the mammalian cyclosporin-bindingprotein cyclophilin. Thus, cyclosporins may mediate someof their effects through cis-trans isomerizations with reactingmolecules.While there are numerous descriptions of the use of these

compounds in clinical studies, the microbiological aspects ofcyclosporin production have been less well documented.The initial description of cyclosporins A and C as metabo-lites of T. polysporum and the taxonomy, fermentationconditions, isolation, characterization, and antimicrobial ac-tivity of these compounds were done by Dreyfuss et al. (9).Later it was shown that by incorporating various aminoacids into the growth medium, synthesis could be directedtowards specific cyclosporins (14). It was also noted briefly(10) that cyclosporin A could be produced in an airlift reactorby carrageenan-entrapped T. inflatum to levels similar tothose of free cells in shake-flask culture. An in-depth studyof the effects of different carbon and nitrogen sources oncyclosporin A production by Agathos et al. (2) indicated thatsorbose was the carbon source of choice for the wild-type T.

* Corresponding author.t Present address: Alberta Environmental Research, Vegreville,

Alberta, Canada TOB 4LO.t Present address: Alberta Research Council, Edmonton, Canada

T6H 5X2.

inflatum ATCC 34921 but that maltose was preferred by theirmutant M6 strain (1). Recently, directed synthesis of cyclo-sporins by using valine and leucine has been shown in aglucose-adapted wild-type strain (16), and this group alsodescribed cyclosporin production by the fungus immobilizedin porous celite beads (7). The enzymatic synthesis ofcyclosporins appears to be mediated by a large, multifunc-tional enzyme (3, 23), which has been shown to be capable ofsynthesizing new cyclosporins in vitro (15).Our interest in cyclosporin production began when we

were unable to obtain reproducible levels of cyclosporinproduction with T. inflatum NRRL 8044. This study wasundertaken to determine the strain and species specificity ofcyclosporin production and the factors contributing to thevariability of production and to establish a reproduciblemethod for obtaining cyclosporin.

MATERIALS AND METHODSOrganisms. All fungal strains used in this study were

obtained from the University of Alberta Microfungus Col-lection and Herbarium (UAMH), Devonian Gardens, Uni-versity of Alberta, courtesy of Lynne Sigler, curator. Theyare designated by their UAMH accession numbers. Thestrains found to produce cyclosporins and their origins areshown in Table 1. All other Tolypocladium species andstrains listed in the UAMH culture catalog were examinedand found to be nonproductive. Cultures were grown andmaintained on malt extract (2%)-yeast extract (0.4%) agarplates, grown at 27°C, and stored at 4°C. Spores from theseagar plates were used as inocula for the experiments sum-marized in Table 1. All other cyclosporin production datawere obtained by using spore stocks from single-sporeisolates of the more active strains, as described below.These were prepared in sterile 20% glycerol at 108spores ml-1 and were maintained at -75°C.Growth in submerged culture. Fungal cultures were rou-

tinely grown in 100 ml of medium in a 500-ml Erlenmeyerflask on a rotary shaker at 200 rpm and at 27°C. Spores (108)from glycerol stocks were inoculated into preculture mediaand were grown for 72 h. This mixture of hyphal clumps andspores was then homogenized for 10 s in a Sorvall Omni-mixer at full speed and used as the inoculum for production

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TABLE 1. Cyclosporin production by strains of T. niveum (Rostrup) Bissett (syn. T. inflatum)

UAMH accession Oiiandecitoofsrn"Cyclosporin producednumber ~~~~~~~~Ofiginand description of strain' (m ie-)bnumber (mg liter-)

2472 Water sample; Whitehorse, N.W.T.; J. W. Carmichael; 15-05-65 101 ± 612880 Soil under Pinus contorta, C horizon; Kananaskis, Alberta; P. Widden, 11-67/G.C. Bhatt 34 ± 41

alpha 77 (=DAOM 167132) cyclosporin A4002 Muskeg soil; Tuktoyatuk, N.W.T.; S. Davies (H-46-F), 1976/S. Davies 46 utilization of 123 ± 75

aliphatic hydrocarbons4553 Soil; Hardanger Vidda, Norway; J. Hansen, 1974/B. Foster (=NRRL 8044=ATCC 32 ± 27

34921=CMI 187376?=UAMH 4740) cyclosporin A and C in submerged culture (9)4594 (N) washed organic particle; alpine meadow; 2530 m, Mt. Allen, Kananaskis, Alberta; 45 ± 38

J. Bissett, 07-05-69/M. Goettel (=DAOM 167322=ATCC 42437)4740 Soil; Hardanger Vidda, Norway; J. Hansen, 1974/J. Vederas (=ATCC 34921=NRRL 12 ± 11

8044=CMI 187376?=UAMH 4553) cyclosporins A, B, C, D, E4828 (T. inflatum) humus alpine soil; Obergurge Oetztal, Austria; W. Gams, 1959/B. Foster 60 ± 35

(=DAOM 64352=CBS 824.70)4900 Mite surface Mycobates sp.; Frobisher Bay, Baffin Island; V. Behan (C), 1976/B. Foster 15 ± 8

(=DAOM 160594)4901 Humified organic material under spruce-fir forest; Luscar, Alberta; S. Visser, 08-76/B. Foster 24 ± 12

(=DAOM 167175)

a Reprinted with permission from the UAMH Culture Collection Catalogue, 1986.b Total cyclosporin (A + B + C) after 10 to 15 days of growth, mean ± standard deviation (n = 6).

flasks at a level of 1 to 10%, depending on the experiment.Production flasks were grown for 12 or 15 days. Highcyclosporin production was obtained by using the mediumdescribed by Agathos et al. (2), which consisted of sorbose(2%), vitamin assay Casamino Acids (1%; Difco Laborato-ries, no. 0288-01-2), KH2PO4 (1%), and KCI (0.5%).

Extraction and quantitation of cyclosporins. Since thecultural morphology after 10 to 15 days of growth wasvariable and often clumpy, cultures were homogenized for10 s and reconstituted to 100 ml with water before sampling.A sample, usually 10 ml, was added to an equal volume ofethyl acetate in a 65-ml medicine bottle, stoppered tightly,and extracted overnight on a reciprocating shaker. Theresulting mixture was centrifuged at low speed (2,500 x g, 10min) to break the emulsion. The organic phase was removedand dried over anhydrous sodium sulfate, and a 1-ml samplewas evaporated to dryness. This sample was dissolved inhigh-pressure liquid chromatography (HPLC) grade acetoni-trile, and 10 ,ul of an appropriate dilution was analyzed byHPLC. Separation and quantitation of cyclosporins wasdone by using a Waters HPLC system: model 501 pump,U6K injector, temperature control module and oven, and amodel 441 absorbance detector at 214 nm attached to aHewlett-Pack,ard 3392A integrator. Separation was per-formed on a Brownlee RP-8 reverse-phase column with anRP-8 NewGuard column. Samples were eluted isocraticallyat 72°C with an acetonitrile-methanol-water (47:20:33) mo-bile-phase run at 2 ml. min-1. A calibration curve wasconstructed by using cyclosporin A, and the slope wasdetermined as 0.166 ,ug of cyclosporin- 106 area units.Cyclosporin A standards were run with each batch ofsamples. A typical experimental chromatogram is shown inFig. 1, in which the relative peak areas of cyclosporins A, B,and C were approximately 7:1:2 for T. niveum grown onunsupplemented medium. All data are reported as the sumtotal of cyclosporins A, B, and C.

Bioassays. Filter disks (10 mm) were impregnated withsolutions of cyclosporins in acetonitrile, dried, and placed onmalt-yeast extract plates. An Aspergillus niger plate wasinverted and tapped lightly to inoculate with spores. Theplate was then incubated at 27°C. A. niger grew as a lawnexcept where inhibited around the filter disks. This assaywas not calibrated for quantitative determinations but was

used only to demonstrate the antifungal activity of thecyclosporins produced by these strains of T. niveum.

Analytical methods. Mycelial dry-weight determinationswere made by filtering 10 ml of culture homogenate throughmembrane filters (HA; Millipore Corp.) and drying overnightat 70°C. Assays of the culture medium components weredetermined on the cell-free supernatants. Sorbose contentwas determined by the method of Dische (8), and the aminoacid content was determined by the fluorodinitrobenzenemethod of Lowry (17). Statistical analysis of data was done

FIG. 1. Chromatogram from HPLC analysis of T. niveumUAMH 2472 metabolites. A T. niveum culture was homogenized,and a sample was extracted with an equal volume of ethyl acetate.A volume of the organic phase was dried, and the residue wasdissolved in half the volume of HPLC-grade acetonitrile. A 10-,ulportion of this solution was injected into the HPLC under thestandard isocratic conditions described in Materials and Methods.Retention times (minutes): cyclosporin C, 3.59; cyclosporin B, 4.02;cyclosporin A, 4.90. Relative peak areas of cyclosporins A:B:Cwere approximately 7:1:2.

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CYCLOSPORINS FROM TOLYPOCLADIUM NIVEUM 123

by using the APL-Mac Duncan's Multiple Range Test (AppleMacIntosh); all tests were done at the 95% confidence level.

Chemicals. All chemicals used were of analytical gradeexcept for solvents, which were HPLC grade. Biochemicalswere obtained from Sigma Chemical Co., St. Louis, Mo.,and growth medium nitrogen bases were from Difco Labo-ratories, Detroit, Mich. Authentic cyclosporin A was kindlyprovided by Sandoz Ltd., Montreal, Quebec, Canada, cour-tesy of G. F. Murphy.

RESULTS

Survey of fungal cultures. The origin of the strains of T.niveum that produced cyclosporins is shown in Table 1. Thistable also includes the range of levels of production in threeseparate early experiments, using one, two, and three flasks,respectively, in each of the experiments. The spore inoculafor these experiments came directly from agar plates, andthe methodology used to extract and quantitate cyclosporinswas not standardized. This resulted in wide fluctuations inproduction for individual strains, as shown by the deviationof the data. Four strains, 2472, 4002, 4594, and 4828,produced more cyclosporin than did strains 4553 and 4740,both of which were lodged with the collection as NRRL8044. The reasons for the different production levels forstrains 4553 and 4740 are unclear, but they were consistentlydifferent. No other Tolypocladium species obtained from theUAMH collection produced cyclosporins, including T.niveum UAMH 4903, nine strains of Tolypocladium cylin-drosporum, two strains of Tolypocladium microsporum, twostrains of Tolypocladium nubicola, and strains of Tolypocla-dium geodes, Tolypocladium tundrense, Tolypocladium ex-

tinguens, and Tolypocladium balanoides. An Indian strain ofT. cylindrosporum has been reported to produce cyclospor-ins after 3 weeks of growth (13), but this was not a charac-teristic of the nine strains tested here.

Single-spore isolates. In their original methodology, Drey-fuss et al. (9) used a single-spore isolate for productionstudies. Taking a similar approach, 20 single-spore isolatesof the four most productive strains were compared. Wideranges in cyclosporin production were observed: T. niveumUAMH 2472 ranged from 40 to 120 mg of cyclosporin.liter-1, with a mean of 72 mg. liter-'; UAMH 2880 rangedfrom 20 to 119, with a mean of 55 mg. liter-'; UAMH 4002ranged from 41 to 100, mean 62 mg. liter-1; and UAMH 4828ranged from 6 to 85, with a mean of 45 mg- liter-1. Sporestocks of the highest-producing single-spore isolate of eachstrain were prepared, stored, and used for all further studies.These stocks have been deposited with the UAMH. The mostactive strain, UAMH 2472, was chosen for future studies.

Identification of T. niveum metabolites as cyclosporins. Toconfirm that the fungal metabolites under investigation werecyclosporins, three tests were done. First, by using HPLCanalysis, authentic cyclosporin A was found to cochromato-graph with the major peak tentatively identified by its elutiontime. Since no authentic samples of cyclosporins B and Cwere available, samples were extracted from large-scaleshake-flask cultures and purified by using silica gel andSephadex LH-20 chromatography as described by Dreyfusset al. (9). The experimental cyclosporin A was eluted as a

single compound, but each of the other two samples was

contaminated by a trace of the other. The identity ofexperimental cyclosporin A (Mw, 1,202.6) was confirmed bymass spectroscopy to have a molecular weight of 1,203 andthe same ion fractionation pattern as the authentic peptide.The sample presumed to be mainly cyclosporin B (Mw,

TABLE 2. Effect of inoculum size on preculture morphology andcyclosporin production by T. niveum UAMH 2472a

Spore Total cyclosporin Mycelial dry wt Mycelialinoculum production (g- liter-' + SD) morphology

102 186 ± 16 7.9 ± 1.5 Pellet103 84 ± 18 5.6 ± 0.4 Pellet104 134 ± 44 8.8 ± 1.1 Pellet105 202 ± 21 7.6 + 0.1 Filamentous106 207 ± 51 8.9 ± 0.2 Filamentous107 229 ± 28 11.2 ± 0.1 Filamentous

Glycerol spore stocks were serially diluted in 50 mM phosphate buffer, pH5.5, and inoculated into triplicate flasks containing 100 ml of sorbose (2%)-vitamin assay Casamino Acids (1%) medium. Flasks were grown for 14 days,at which time all flasks exhibited good mycelial growth.

1,188.6) showed a molecular weight of 1,189, while that ofcyclosporin C (Mw, 1,218.6) was 1,219, both samples corre-sponding to the published structures (1) and their expectedelution sequence from a reverse-phase HPLC column on thebasis of their hydrophobicity. All three isolated cyclosporinsinhibited the growth and sporulation of A. niger on agarplates.Inoculum and preculture optimization. One of the most

variable parts of the procedure is the preparation of thepreculture with which the production culture is to be inocu-lated. Some variation has been reported in the spore inoculaand the preculture media that have been used; therefore, itwas decided to investigate both. The effect of the sporedensity of the preculture on cyclosporin and mycelial yieldsis shown in Table 2. After 14 days of growth, the higherspore inocula generally resulted in higher cyclosporin pro-duction and a characteristic filamentous mycelial morphol-ogy. Low spore inocula gave rise to a more clumpy mor-phology and lower cyclosporin production. The same trendwas observed in production flasks, that is, precultures fromhigh spore inocula gave high cyclosporin titers in productionflasks (Fig. 2). The data in this figure are from a singletriplicate-flask experiment, but each experiment was re-peated either twice or three times to ensure reliability.Repeated experiments confirmed that for consistently highcyclosporin production, a spore inoculum of at least 104spores. ml-' should be used for precultures. The inoculumbiomass from preculture to production culture was alsoinvestigated. In two separate experiments, both done withtriplicate flasks for each biomass point, inoculum volumes of1 to 20%, corresponding to biomass ranges from 6.1 to 123mg, and volumes of 1 to 10%, corresponding to from 8.6 to86 mg of dry weight of mycelium per flask, were examined.Only the highest and lowest inoculum sizes gave significantlydifferent, lower cyclosporin production; therefore, a 5 to10% inoculum representing 50 to 70 mg (dry weight) ofmycelium was used as the standard. The age of the precul-ture inoculum was another variable which was examined:precultures were grown from 24 to 150 h before use as aninoculum. Again, while there was some variation in the dataobtained, they were statistically identical, and a 72-h precul-ture time was chosen for routine experiments. The finalaspect of inoculum culture development was to compare twopreculture media, one complex of malt extract (2%)-yeastextract (2%) and one of semisynthetic sorbose (2%)-vitaminassay Casamino Acids (1%). After 72 h of growth, eachpreculture was divided, one-half was washed three timeswith 50 mM potassium phosphate buffer, pH 5, and the otherhalf was not washed. Both washed and unwashed precul-

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FIG. 2. Effect of spore inoculum size on final cyclosporin levels produced by T. niveum UAMH 2472 in production medium. Precultureflasks containing 100 ml of sorbose (2%)-vitamin assay Casamino Acids (1%) medium were inoculated with different numbers of spores of T.niveum UAMH 2472 from glycerol stocks. These precultures were incubated 14 days to ensure complete growth in each flask, and then 10ml was used as inoculum for sets of four production flasks of the previously described medium. After 14 days of growth, cultures werehomogenized and analyzed for total cyclosporin content and mycelial dry weight. Cl, Total cyclosporin production; *, mycelial dry weight.

tures were then used to inoculate sorbose-Casamino Acidsproduction media at 1, 2, 5, and 10% (wt/vol) inoculumlevels. Results showed that there was little difference incyclosporin production between the different inoculum lev-els or between the washed and unwashed inocula. However,both the volumetric and the specific production of thesorbose-grown precultures were three- to fourfold higherthan the malt-yeast extract preculture.Medium development. A variety of carbon sources have

been shown to support cyclosporin production by T. in-flatum ATCC 34921 (2). Of these 22 carbon sources, sorbosesupported both the highest volumetric and specific cyclo-sporin A production, although somewhat erratically. It wasof interest to determine whether the UAMH strains of T.niveum responded equally well to sorbose or whether theyshowed a preference for maltose (1) or glucose (11). The fivemost productive strains were therefore grown on these threecarbon sources and on fructose (like sorbose, a 2-ketohex-ose), and the results (Table 3) showed that for four of the fivestrains, sorbose was the most productive carbon source. Allfour carbon sources supported good growth (data not pre-

TABLE 3. Cyclosporin production by five strains ofT. inflatum grown on four carbon sources0

UAMH Total cyclosporin production (mg liter-' SD)strain Sorbose Fructose Glucose Maltose

2472 145.0 + 17.8 68.0 0.8 63.2 + 5.3 71.9 ± 14.02880 125.0 44.2 68.6 + 3.2 22.0 + 1.7 60.5 5.54002 130.0 + 14.4 62.2 9.7 60.1 5.9 59.4 6.34553 10.5 + 1.2 32.6 7.9 79.1 + 15.7 55.0 11.94828 60.0 5.1 22.3 + 5.5 22.5 + 2.0 17.8 2.6

a Inoculum preparation was as, follows: i0o spores from washed sporestocks were inoculated into 100 ml of sorbose (2%o)-vitamin assay CasaminoAcids (1%) medium and the precultures were grown for 72 h. Cultures werehomogenized for 10 s, and 10 ml was inoculated into triplicate flasks of eachcarbon source. Growth was for 14 days, and then cultures were homogenizedand made up to 100 ml with water before extraction.

sented). These data confirmed the earlier indications that2472 is the most productive strain and sorbose is the carbonsource of choice (2). A wider selection of carbon sourceswas examined by using only T. niveum UAMH 2472, withtriplicate flasks, and the following data were obtained, inmilligrams of cyclosporin. liter1 + the standard deviation:sorbose, 140 ± 21; glucose, 132 ± 9.5; xylose, 116 ± 4.4;glycerol, 107 ± 5.4; ribose, 94 ± 5.9; galactose, 93 ± 2.6;fructose, 64 ± 1.7; maltose, 53 ± 4.6; sucrose, 27 ± 2.8;lactose, 19 ± 1.5; cellulose 17 ± 2.4; and inulin, 6 + 0.7.A variety of complex nitrogen sources were then com-

pared for their support of cyclosporin production, usingsorbose (2%) as the carbon source. The results are shown inTable 4. Vitamin assay Casamino Acids was arbitrarilychosen as the most consistent nitrogen source, althoughCasitone or peptone gave comparable cyclosporin yields.However, the ethyl acetate extracts of cultures grown onvitamin assay Casamino Acids separated cleanly from theaqueous phase and exhibited a minimum number of extra-neous HPLC peaks. Both biomass and cyclosporin yieldincreased with the concentration of complex nitrogen source

TABLE 4. Cyclosporin production by T. niveum UAMH 2472 ona variety of nitrogen sources'

Nitrogen source Final Mycelial dry wt Cyclosporin production(1%, wt/vol) pH (g- liter1') (mg liter-1 ± SD)

Negative control 4.5 1.1 23 ± 2Casamino Acids 5.1 1.8 65 ± 16Vitamin assay 4.4 13.7 156 + 6Casamino Acids

Bacto-Casitone 5.0 12.6 121 ± 40Bacto-Peptone 5.1 8.6 119 + 31Tryptone 5.6 9.2 111 ± 18

a Inoculum was prepared as described in the legend to Table 3. Inoculationsof 10 ml were made into groups of three flasks of 2% sorbose-1% nitrogensource media. Note that the negative control (no added nitrogen source) andCasamino Acids were only done in duplicate. The fermentation period was 12days.

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TABLE 5. Directed synthesis of cyclosporins in five T. niveum strains

Strain; precursor Mycelial dry wt Total cyclosporin Cyclosporins as % of total.(8 g liter-') (g liter-') (mg liter-' + SD) A B C G-

2472Control 7.4 ± 0.17 79 ± 10 61 14 25 0DL-AbUb 8.7 ± 0.12 115 ± 8 69 5 26 0L-Ala 8.7 ± 0.21 87 ± 11 66 15 19 0L-Val 9.5 0.21 135 12 61 0 9 0DL-Nvab 9.8 0.60 135 ± 11 24 0 6 70

2880Control 7.9 ± 0.15 52 ± 3 77 0 23 0DL-Abu 9.4 ± 0.12 30 ± 2 67 0 33 0L-Ala 9.4 ± 0.17 61 ± 3 75 10 15 0L-Val 10.2 ± 1.3 89 ± 13 85 0 15 0DL-Nva 10.9 ± 0.44 75 ± 7 19 0 12 69




a Tentatively identified.b DL-AbU, DL-cX-aminobutyrate; DL-Nva, DL-norvaline.

up to 1% (wt/vol), but no significant increase in yield wasobserved beyond this concentration. The presence of addednitrate (11) was found to reduce rather than stimulate cyclo-sporin yields in- our experiments. Increasing or decreasingthe phosphate or chloride levels had little effect on produc-tion.

Directed synthesis. A particularly interesting aspect of thestudy of Kobel and Traber (14) was that by including specificamino acids in the growth medium, the organism could bedirected towards production of a specific product. To deter-mine whether this principle was widely applicable, the fiveT. niveum strains initially found to produce high levels ofcyclosporins were grown in the presence of amino acidsupplements. Groups of three flasks were used for eachstrain and supplement, and the results are shown in Table 5.The results were somewhat disappointing in that the abso-lute values are lower than those reported in Tables 1 to 4,and, while some data reflected very closely an earlier pre-liminary experiment, other data, particularly the DL-a-ami-nobutyrate, were quite different. Clearly, cyclosporin syn-thesis can be directed in all of the strains investigated, butresults indicate that supplementation with amino acids doesnot always increase the total synthesis.Time course of cyclosporin production. One of the prob-

lems of sampling single flasks of a filamentous fungus isobtaining representative samples from a clumpy culture. Thegrowth curve shown in Fig. 3 was obtained from the analysis

of sets of three flasks of T. niveum UAMH 2472 harvestedeach day for 18 days. The data points show some scatter butgenerally follow the same pattern as that described byAgathos et al. (2) for T. inflatum ATCC 34921 grown onsorbose and, to a lesser extent, that of Dreyfuss et al. (9).Rapid growth over the first 4 days correlated with a declinein the carbon and nitrogen content of the medium. Slowergrowth occurred then up to day 10, which coincided with themaximum cyclosporin yield and carbon source depletion.Attempts to improve cyclosporin yields by feeding addi-tional carbon source at this point have so far been unsuc-cessful with this strain.

DISCUSSION

The microbial production of cyclosporins has receivedlittle attention in the research literature, and developmentsin cyclosporin fermentation technology have been few (1, 2,7, 9, 11, 14-16). The studies described here resulted fromobservations of low and inconsistent cyclosporin productionfrom the strain T. inflatum NRRL 8044, originally describedand deposited by the Sandoz group. Differences in the fungalstrains, media, and growth conditions used by other work-ers, coupled with a lack of published data on consistency andreproducibility of production, stimulated this approach. Wehave shown that many of the strains of T. niveum on depositat the University of Alberta Microfungus Collection pro-

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25

00 10 12 14 16 18 20

Time (days)

FIG. 3. Time course of cyclosporin production by T. niveum UAMH 2472 in shake flasks. Five preculture flasks containing 100 ml ofsorbose (2%)-Casamino Acids (1%) medium were each inoculated with 108 spores of T. niveum UAMH 2472 from glycerol stocks. Theseflasks were incubated for 72 h and then pooled and diluted to 560 ml with water. A total of 54 flasks, each containing 100 ml of the previouslydescribed medium, were inoculated with 10 ml of preculture. Every day for 18 days, groups of three flasks were removed and the cultureswere homogenized and analyzed. *, Total cyclosporin production; 1, mycelial dry weight; O, sorbose; <, vitamin assay Casamino Acids.

duced more cyclosporin than did the NRRL strain, whichwas the parent of two UAMH cultures. This was observedconsistently and with different media. Thus the NRRL strainmay well be a poor starting point for the study of variousaspects of cyclosporin production. Only one UAMH T.niveum strain, 4903, did not produce cyclosporins, and noneof the other seven Tolypocladium spp., 17 strains in all, wereable to do so.The initial screening was done with 10 strains, using agar

plates as the source of inocula and making changes to themethods in each of the three experiments. These data werevery erratic, changing by as much as 50% between experi-ments. The inherent biological variation was shown whencultures were grown from single-spore isolates, and usingspore stocks from the highest-producing single-spore iso-lates reduced the variation in production considerably. Thechoice of T. niveum UAMH 2472 as the strain to study indetail was based initially on the data obtained from thesingle-spore isolates. Equally possible, strain 2880 mighthave been chosen, or even 4002, although the data presentedin Table 3 confirm 2472 as the strain of choice.

It is perhaps not surprising that the preculture, whenallowed to grow for 14 days, shows slightly higher totalcyclosporin production (Table 2) than the production culture(Fig. 2), although in this study they are statistically equiva-lent. The decline in productivity of fungal metabolite pro-duction as a culture is transferred into fresh medium is aproblem encountered by many pharmaceutical companies

when scaling up to production volumes. We have no data toindicate how many transfers of vegetative cells are requiredbefore cyclosporin production is significantly reduced.

Particular attention was paid to the method required toobtain reproducible data. The inclination of these fungi togrow in clumps, or even as 2- to 3-mm pellets, necessitateda homogenization step prior to sampling. Since such dis-persal methods might disturb the relationship of morpholog-ical events to secondary metabolite formation, homogeniz-ing, sampling, and returning the homogenate to the shakerwas not used in analyzing the growth curve. Instead, threeseparate flasks were harvested for each day, processed, anddiscarded.

Sequential samples were not taken from the same flask.Even when all precautions were taken, there were stilloccasions when anomalous results arose, but this is stillunder investigation and also is not unique in working withfilamentous fungi.The relationship between spore inoculum density and

culture morphology is well established (18). The resultsshowing that higher spore densities are required for highcyclosporin production confirm the methodology describedby Dreyfuss et al. (9) and argue in favor of a preciseinoculum. The carbon source described by Agathos et al. (2)as optimal for T. inflatum ATCC 34921 was also mosteffective for most of the strains investigated in this study. Ofthe complex nitrogen sources tested, vitamin assayCasamino Acids produced the most consistent data and the

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cleanest extract. Now that a reproducible methodology hasbeen determined, it is intended that further investigation willbe done to examine more closely the biochemistry of cyclo-sporin formation and its control in these organisms.

ACKNOWLEDGMENTS

We thank Atsumi Hashimoto for technical assistance and LynneSigler of the University of Alberta Microfungus Collection for fungalcultures and advice on taxonomy.

This work was supported by grants from the Natural Sciences andEngineering Research Council of Canada and the Alberta HeritageFund for Medical Research to M.A.P.

LITERATURE CITED1. Agathos, S. N., C. Madhosingh, J. W. Marshall, and J. Lee.

1987. The fungal production of cyclosporine. Ann. N.Y. Acad.Sci. 506:657-662.

2. Agathos, S. N., J. W. Marshall, C. Moraiti, R. Parekh, and C.Madhosingh. 1986. Physiological and genetic factors for processdevelopment of cyclosporin fermentations. J. Ind. Microbiol.1:39-48.

3. Billich, A., and R. Zocher. 1987. Enzymatic synthesis of cyclos-porin A. J. Biol. Chem. 262:17258-17259.

4. Bissett, J. 1983. Notes on Tolypocladium and related genera.Can. J. Bot. 61:1311-1329.

5. Borel, J. F. 1986. Cyclosporin and its future. Prog. Allergy38:9-18.

6. Canon, P. F. 1986. International commission on the taxonomyof fungi (ICTF): name changes in fungi of microbiological,industrial and medical importance, part 2. Microbiol. Sci. 3:285-287.

7. Chun, G.-T., and S. N. Agathos. 1989. Immobilization of Toly-pocladium inflatum spores into celite beads for cyclosporineproduction. J. Biotechnol. 9:237-254.

8. Dische, Z. 1962. Color reactions of hexoses. Methods Carbo-hydr. Chem. 1:488-494.

9. Dreyfuss, M., E. Harri, H. Hoffman, H. Kobel, W. Pache, and H.Tscherter. 1976. Cyclosporin A and C. New metabolites fromTrichoderma polysporum (Link ex Pers.) Rifai. Eur. J. Appl.

Microbiol. 3:125-133.10. Fischer, G., B. Wittman-Liebold, K. Lang, T. Kiefhaber, and

F. X. Schmid. 1989. Cyclophilin and peptidyl-prolyl cis-transisomerase are probably identical proteins. Nature (London)337:476-478.

11. Foster, B. C., R. T. Coutts, F. M. Pasutto, and J. B. Dosseter.1983. Production of cyclosporin A by carrageenan-immobilizedTolypocladium inflatum in an airlift reactor with external loop.Biotechnol. Lett. 5:693-696.

12. Gams, W. 1971. Tolypocladium. Eine Hyphomycetengattungmit geschwollenen Phialiden. Persoonia 6:185-191.

13. Jayaraman, K. S. 1988. Cyclosporin-yielding fungus found.Nature (London) 332:671.

14. Kobel, H., and R. Traber. 1982. Directed biosynthesis ofcyclosporins. Eur. J. Appl. Microbiol. Biotechnol. 19:237-240.

15. Lawen, A., R. Traber, D. Geyl, R. Zocher, and H. Kleinkauf.1989. Cell-free biosynthesis of new cyclosporins. J. Antibiot.42:1283-1289.

16. Lee, J., and S. N. Agathos. 1989. Effect of amino acids on theproduction of cyclosporin A by Tolypocladium inflatum. Bio-technol. Lett. 11:77-82.

17. Lowry, 0. H. 1966. Appearance of free amino groups duringlysis of bacterial cell walls. Methods Enzymol. 8:118-119.

18. Metz, B., and N. W. Kossen. 1977. The growth of molds in theform of pellets. Biotechnol. Bioeng. 19:781-799.

19. Rifai, M. A. 1969. A revision of the genus Trichoderma. Mycol.Papers 116:1-56.

20. Sigler, L., S. P. Frances, and C. Panter. 1987. Culicinomycesbisporalis, a new entomopathogenic hyphomycete from larvaeof the mosquito Aedes kochi. Mycologia 79:493-500.

21. Takahashi, N., T. Hayano, and M. Suzuki. 1989. Peptidyl-prolylcis-trans isomerase is the cyclosporin A-binding protein cyclo-philin. Nature (London) 337:473-475.

22. von Arx, J. A. 1986. Tolypocladium, a synonym of Beauveria.Mycotaxon 25:153-158.

23. Zocher, R., T. Nihira, E. Paul, N. Madry, H. Peeters, H.Kleinkauf, and U. Keller. 1986. Biosynthesis of cyclosporin A:partial purification and properties of a multifunctional enzymefrom Tolypocladium inflatum. Biochemistry 25:550-553.

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production of cyclosporins by tolypocladium niveum strains

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