Transformations of Progesterone by Basidiomycetes

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					                  Transformations of Progesterone by Basidiomycetes
EUNICE C. SCHUYTEMA, MARTHA P. HARGIE, DONALD J. SIEHR,' ILMAR MERITS, JAY R. SCHENCK, MURIEL S.                                    SMITH,
                                        AND ESTIE L. VARNER

                  Department of Biochemistry, Research Division, Abbott Laboratories, North Chicago, Illinois
                                          Received for publication 14 January 1963
                       ABSTRACr                                     TABLE 1. Number and types of basidiomycetes used with progesterone
  SCHUYTEMA, EUNICE C. (Abbott Laboratories, North                               Genus                No. of species   No. of species trans-
                                                                                                        examined       forming progesterone
Chicago, Ill.), MARTHA P. HARGIE, DONALD J. SIEHR,
ILMAR MERITS, JAY R. SCHENCK, MURIEL S. SMITH, AND                  Agaricus ......................         6                   1
ESTIE L. VARNER. Transformations of progesterone by                 Agrocybe......................          5                   1
basidiomycetes. Appl. Microbiol. 11:256-259. 1963.-A                Amanita......................           7                   3
                                                                    Armillaria....................          4                   0
total of 254 basidiomycete cultures have been examined              Auriscalpium .................          1                   0
for their action on progesterone. Of these, 54 showed               Boletinus .....................         2                   1
                                                                    Boletus .......................         6                   4
transformation products by thin-film and gas-liquid                 Calvatia ......................         1                   0
chromatography. The major product formed by eight of                Clavaria......................          4                   0
                                                                    Clitocybe......................        22                   4
these organisms acting on progesterone has been isolated            Clitopilus.....................         1                   0
and identified.                                                     Collybia ......................        18                   1
                                                                    Coprinus......................          4                   2
                                                                    Cortinarius ...................         2                   1
                                                                    Crepidotus....................           1                  0
   An early report in the literature of the bio-oxygenation         Cystoderma ...................           1                  0
of progesterone by mushrooms (Roland and Weiner, 1955)              Dacryomyces ..................           1                  0
                                                                    Dermoloma....................            1                  1
led us to investigate the activity of a large number of             Dictyophora ...................          1                  0
basidiomycetes from our collection on the progesterone              Flammula ....................            1                  0
                                                                    Galerina......................           3                  0
molecule. It was hoped that since basidiomycetes are more           Gymnopilus ...................          5                   2
highly differentiated organisms their transforming capacity         Hebeloma.....................           2                   0
                                                                    Hericeum.....................           2                   0
i.e., their complement of enzymes, might differ markedly            Hygrophorus ...............             3                   1
from that of the lower fungi and bacteria used previously           Hypholoma....................           1                   0
                                                                    Hypomyces ...................           1                   0
for the transformation of the steroid nucleus.                      Kuehneromyces ................          3                   0
                                                                    Laccaria......................          1                   0
                 MATERIALS AND METHODS                              Lactarius.....................          6                   2
                                                                                                                                0
                                                                    Lentinellus ...................         1
                                                                                                                                1
   Cultural methods. Cultures were isolated from naturally          Lentinus......................          3
                                                                                                                                1
                                                                    Lentodium....................           1
occurring basidiomycetes (identified by A. H. Smith,                Lepiota.......................          1                   1
University of Michigan Herbarium) either by tissue culture          Lepista .......................        11
                                                                                                            7
                                                                                                                                1
                                                                                                                                1
of the sporophore or by germination of spore collections.           Leucopaxillus .................                             0
                                                                    Lycoperdon ...................          5
Cultures were maintained on malt agar slants, under                 Lyophyllum...................           2                   1
                                                                    Marasmius ...................          16                   1
mineral oil, in the dark, at 4 C.                                   Melanoleuca...................          1                   0
   Inoculum was produced in 250-ml Erlenmeyer flasks                Mycena.......................          19                   1
                                                                                                                                1
                                                                    Naematoloma..................           7
containing 50 ml of 4 % malt extract broth. Pieces of               Naucoria .....................          1                   1
mycelium were allowed to grow for 5 days on a rotary                Panellus ......................         1                   1
                                                                    Panaeolus .....................         1                   1
shaker at 25 C. The mycelium was transferred by means               Panus........................           4                   0
of a 30-ml syringe equipped with an 18-gauge needle with            Paxillus ......................         3                   2
                                                                    Philloporus ...................         2                   0
the point cut off. Mycelial clumps were broken up by                Pholiota.........                       8                   1
several passages through the syringe. A 2-ml portion of the         Pleurotus .....................         8                   0
                                                                    Pluteus.......................          2                   1
mycelial suspension was used for inoculum.                          Polyporus ....................         18                   6
    To 24-hr broth cultures, which had been incubated on            Psilocybe .....................         1                   1
                                                                                                                                0
                                                                    Rhodophyllus ..................         2
a shaker at 25 C, were added 30 mg of the steroid to be             Russula ......................          2                   1
transformed in 1 ml of methanol. After addition of the              Schizophyllum .................         1                   0
                                                                    Stropharia ....................         1                   1
steroid, the cultures were reincubated on a shaker for 48 hr.       Tricholoma..                            2                   1
                                                                    Tricholomopsis ....                     3                   0
   1 Present address: Department of Chemical Engineering and                                                1                   1
                                                                    Tubaria.......................                              1
Chemistry, University of Missouri School of Mines and Metal-        Xeromphalina.................           3
lurgy, Rolla.
                                                              256
VOL. 11, 1963                       ACTIVITY OF BASIDIOMYCETES ON PROGESTERONE                                                                               257
                                        TABLE 2. Relative percentage of transformation products
                                                                                                         Retention times*
                     Culture name                    Culture no.
                                                                   0.55   0.70 0.85   1.0   1.08   1.3    1.5           1
                                                                                                                      1.7 .8       1.95 2.12 2.25 2.42 2.55 2.70

Lactarius chysorrheus ..........................     F-31                  5    30    60            5
L. volemus ...................................       SS-15                             5                  10                       40        10   35
Russula delicans ..............................      C-173                            30     9     10     50                                  1
Hygrophorus conicus ..........................       C-219                            T                                            20             70
Pluteus granularis .............................     C-123                            T                  100
Amanita muscaria .........................           F-6                              T                                            20   10        70
A. muscaria ...................................      H-26                       10    90
A. porphyria ..................................      H-104                      10    50                        40
Lepiota naucina ...............................      G-59                             10                        30                                60
Agaricus edulis ...............................      G-98                             60                                           10         5   25
Lentinus vulpinus .............................      C-137                       5    25           30     30    10
Lentodium squamosum.........................         G-17                 10    40    40                        10
Leucopaxillus paradoxus     ......................   F-55                             T                                            20   10        70
Panellus stypticus   .............................   C-224                      15    70           15
Xeromphalina tenuipes ........................       SS-6                             29                    1     5                 5             60
Clitocybe adirondackensis .......................    SS-43                 3   85     12
Clitocybe "C"..................................
             .                                       G-8                              18            2           30
C. clavipes ....................................     G-107                            25                                           20             40         15
C. odora ......................................      H-21                  1     1    93                          5
Lyophyllum aggregatum .......................        SS-60                             1                               29                         70
Mycena strobilinoides .........................      SS-75                 5     5    90
Collybia dryophia .............................      C-59                             T                  100
Marasmius siccus ..............................      SS-21                            40                               60
Lepiota molybdites .............................     G-19                              8                    4                      18             70
Dermoloma "SP" ................................      F-27                             T                                            20   10        70
Tricholoma "SP"  ...............................     G-88                             10                    5   10            10                  65
Paxillus involutus .............................     H-19                              3                    2                      30             65
P. vernalis ....................................     H-25                  1   44     55
Tubaria conspersa .............................      SS-32                            90                    5                                      3
Naucoria confragosa ...........................      C-172          2                 T                     8                           45        45
Phcliota squarrosoides .........................     H-140                            85                        15
Gymnophilus junenius .........................       C-142                     25     75
Gymnopilus # 2 ...............................       G-69                                                   5                      15             80
Cortinarius evernius ...........................     C-351                10   50     35           T       T     T
Agrocybe acericola .............................     SS-14                      3     85                    3         7                                  2
Stropharia nordmanii ..........................      SS-74                      3     47                        50
Naematoloma sublateratium ....................       H-6                       10     90
Psilocybe caerulipes   ...........................   C-236          5                 13                  25    40                                           17
Coprinus atromentarius ........................      G-75                             10                                           20             70
C. sobiliferus ..................................    G-63                             70                                           15                  15
Panaeolus papilionaceus .......................      G-60                 T     15    50                    2   25                                 8
Boletus acidus .................................     H-33                             10                                           20             70
B. americanus .................................      G-119                            80                        20
B. uteus ......................................      H-1l                              1                    1                       8             80         10
Boletinus pictus ...............................     F-12                             T                                            20   10        70
Polyporus brumalis ...........................       G-67                  5    10    50           30            5
P. caeruleoporus ..............................      H-126                      25    25                    5   30                           15
P. cinnabarinus ...............................      G-90                  3     7    70                        20
P. conchifer ...................................     C-255                       5    80                                  5                       10
P. radicata ....................................     H-139                 5    65    30
P. squamosus .................................       C-180                10    20    70
P. sulfureus ...................................     C-6                  10          10    50              5             5        20
Mex 25 ....................................          SS-70                       1    50                    4   30                                15
Mex 228 ....................................         SS-50                            15                         5      10                   70
Unidentified ..................................      Z-9                              10                   5            25                        60
Unidentified .......                                 Z-12                             60                  13    13                            7    7
   *
    The figures at the head of the vertical columns represent retention times, relative to progesterone at 1.0. Figures in the columns
represent relative areas under the peaks of the gas chromatograms of the chloroform extracts (see text) compared with standards
using SE-30 column. T = trace.
258                                                       SCHUYTEMA ET AL.                                     APPL. MICROBIOL.
  Analytical methods. The whole culture, mycelium and                 SE-30, polar fluorinate alkyl silicone XF-1, polyether
beer, was extracted three times with chloroform, the                  Pluronic F-68, and polyester neopentyl glycol succinate.
chloroform extract was taken to dryness in vacuo, and the             The quantitative responses to various molecular species
residue was taken up in chloroform. This material was                 (at 500 v across the ionization chamber; Lovelock, 1958;
examined by the thin-layer chromatographic technique                  Lovelock, James, and Piper, 1959) and their relative
(Van Dam, DeKleuver, and DeHeus, 1960; Privett and                    retention times (progesterone = 1.00) were determined
Blank, 1961). When there was any indication of trans-                 with known steroids. This enabled us to get a semiquanti-
formation on the silica gel plate, the extracts were exam-            tative estimate of the fermentation products.
ined by gas-liquid chromatography. A Barber Coleman                      Isolation and identification. Some of the transformation
model 10 gas chromatograph with a Sr'O argon ionization               products were isolated from 7-liter fermentation batches
detector and a glass column (180 cm X 6 mm) at 220 C                  by extraction with chloroform, chromatography on silica
was used. Flash heater temperature was kept at 250 to                 gel, and crystallization. These were identified by gas
300 C. The solid-support, Chromosorb W, 60-80 mesh was                chromatography, thin-film chromatography, infrared and
siliconized (Howard and Martin, 1950) and coated with                 ultraviolet spectra, and elemental analysis.
0.5 to 1 % of one of the stationary phases. Four stationary
phases were employed: nonpolar methyl silicone gum                                              RESULTS
                                                                         A total of 254 cultures were screened for their ability to
TABLE 3. Comparison of basidiomycete progesterone transformation      transform progesterone (see Table 1). Of these cultures, 56
                 products with known steroids                         showed one or more transformation products, in reasonable
Retention No. of   Transformation           Sadr      tris            yield, when the extract was examined by thin-film chroma-
 time on   cul-b      products'             Standard steroidsd
 SE- 30o1 turesb      rdCS                                            tography.
                                                                         Table 2 shows the retention time and relative percentage
0.55         2                                                        of progesterone and its transformation products found
0.70         8                   Pregnane-3,20-dione, 4-andros-       when these extracts were examined by gas-liquid chroma-
                                   tene-3, 17-dione                   tography. No clear-cut correlation between classification
0.85        14     Allopregnane- Pregnenolone, testosterone, cor-
                     3, 20-dione   tisone, 4-androstene-3, 11,17-     and transformation products is obvious. Therefore, it
                                   trione, 6,6-hydroxy-4-andros-      would appear that morphologically closely related basidio-
                                   tene-3, 17-dione, progesterone,    mycetes do not necessarily contain the same complement
                                   16a-hydroxyprogesterone, 6#,       of enzymes.
                                   17a , 21-trihydroxy-11-keto-          Table 3 indicates the possible identity of the steroid
                                   progesterone
1.08         2                   1,4-Pregnadiene-3, 20-dione, cor-    transformation products, based on a comparison of reten-
                                   tisol                              tion times with standard known steroids. Some retention
1.3          3                                                        times are common to several basidiomycete cultures.
1.4-1.5     16                      17a-Hydroxyprogesterone, 14a-        Cultures and identifiable transformation products
                                     hydroxyprogesterone              isolated from them (shown in parentheses) follow: Corti-
1.52         2     Testololac-      12a-Hydroxyprogesterone
                     tone                                             narius evernius C-351 (allopregnane-3,20-dione); Plutus
1.7         19                      llci-Hydroxyprogesterone, 116-    granularis C-123 and Collybia dryophila C-59 (testolo-
                                      hydroxyprogesterone,     15a-   lactone), Dermoloma sp. F-27, Hygrophorus conicus C-219,
                                      hydroxyprogesterone, 15,-hy-    Leucopaxillus paradoxus F-55, and Boletus luteus H-1l
                                      droxyprogesterone,     21-hy-   (6(, 1 la-dihydroxyprogesterone); Naucoria confragosa
                                      droxyprogesterone
1.8          6                                                        C-172, (6,3, 17a-dihydroxyprogesterone).
1.95        16                      7a-Hydroxyprogesterone
2.12         5     63,17a-Dihy-                                                               DISCUSSION
                     droxypro-                                           It can be seen from an examination of Table 2 that the
                     gesterone
2.25         6                                                        basidiomycetes possess a large number of enzymes capable
2.42        25     6#,11a-Dihy-                                       of acting on the steroid nucleus, as represented by pro-
                     droxypro-                                        gesterone. However, only a few of these transformation
                     gesterone                                        products are produced in sufficient yields to allow isolation
2.55         1                                                        of the products. All the identified products isolated have
2.70         3
                                                                      been reported before as transformation products from other
     Retention times are relative to progesterone at 1.0.             microorganisms (Peterson et al., 1952; Fried, Thoma, and
     Number of cultures is the number in which these trans-
      b                                                               Klingsberg, 1953; Meister et al., 1953; Schubert, Bohme,
formation products were found.                                        and Horhold, 1961). In fact, it would appear that certain
   c These products were isolated in pure form from some of the
fermentations.                                                        enzymes for modifying the steroid nucleus are very wide-
   d Most of the standard steroids were kindly supplied by E. G.      spread in microorganisms, no matter how highly differ-
Daskalakis of G. D. Searle & Co.                                      entiated they may be.
VOL. 11, 1963                 ACTIVITY OF BASIDIOMYCETES ON PROGESTERONE                                                           259
                       LITERATURE CITED                                  dihydroxyprogesterone and Ila-17a-dihydroxyprogesterone.
                                                                         J. Am. Chem. Soc. 75:416-418.
FRIED, J., R. W. THOMA, AND A. KLINGSBERG. 1953. Oxidation of        PETERSON, D. H., H. C. MURRAY, S. H. EPPSTEIN, L. M. REINEKE,
     steroids by microorganisms. III. Side chain degradation, ring       A. WEINTRAUB, P. D. MEISTER, AND H. M. LEIGH. 1952. Micro-
     D cleavage and dehydrogenation in ring A. J. Am. Chem. Soc.         biological transformation of steroids. I. Introduction of oxy-
    75:5764-5765.                                                        gen at C-11 of progesterone. J. Am. Chem. Soc. 74:5933-5936.
HOWARD, G. A., AND A. J. P. MARTIN. 1950. The separation of the      PRIVETT, 0. S., AND M. L. BLANK. 1961. A new method for the
     C12-C18 fatty acids by reversed-phase partition chromatog-          analysis of component mono, di, and triglycerides. J. Lipid
     raphy. Biochem. J. 46:532-538.                                      Res. 2:37-44.
LOVELOCK, J. E. 1958. A sensitive detector for gas chromatog-        ROLAND, J. F., JR., AND B. A. WEINER. 1955. Bio-oxygenation of
     raphy. J. Chromatog. 1:35-46.                                       progesterone by mushrooms. Science 121:803-804.
LOVELOCK, J. E., A. T. JAMES, AND E. A. PIPER. 1959. A new type      SCHUBERT, K. VON, K.-H. BOHME, AND C. HORHOLD. 1961. Forma-
     of ionization detector for gas chromatography. Ann. N.Y.            tion of aromatization and hydrogenation products from
    Acad. Sci. 72:720-730.                                               progesterone by Mycobacteriurm smegmatis. Z. Naturforsch.
 MIEISTER, P. D., D. H. PETERSON, H. C. MURRAY, G. B. SPERO,             16:595-597.
    S. H. EPPSTEIN, A. WEINTRAUB, L. M. REINEKE, AND H. M.           VAN DAM, M. J. D., G. J. DEKLEUVER, AND J. G. DEHEUS. 1960.
    LEIGH. 1953. Microbiological transformation of steroids. V.          Thin layer chromatography of weakly polar steroids. J.
    The oxygenation of 17a-hydroxyprogesterone to 60, 17a-               Chromatog. 4:26-33.

				
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