FEMS Microbiology Letters 111 (1993) 203-206
© 1993 Federation of European Microbiological Societies 0378-1097/93/$06.00
Published by Elsevier
203
FEMSLE 05539
Screening fungi for the production 0fan inhibitor
of HMG CoA reductase: Production of mevinolin
by the fungi of the genus Pleurotus
Nina G u n d e - C i m e r m a n a, Jo~ica Friedrich a, Aleksa C i m e r m a n a and Neda Beni~ki b
a National Institute o f Chemistry, Ljubljana, Slovenia, and b Pharmaceutical factory Krka, Novo mesto, Slovenia
(Received 21 April 1993; revision received and accepted 10 May 1993)
Abstract: We have tested 380 strains of our Fungal Culture Collection (MZKIBK), for the activity of the inhibitor of HMG CoA
reductase, mevinolin. All fungal strains were cultivated in a two-step submerged fermentation on selected substrates. For the first
screening, samples of broth extracts were tested by TLC and positive results were further evaluated by HPLC analysis. In addition
to already known microbial producers of HMG CoA reductase inhibitors and some newly identified strains having moderate
activity, we found that fungi of the basidiomycetous genus Pleurotus, especially species P. ostreatus, P. saca and P. sapidus, are an
as yet unknown but promising source of the hypocholesterolaemic agent.
Key words: Pleurotus; Mevinolin; Cholesterol; HMG CoA reductase inhibitor; Screening of fungi
Introduction
Mevinolin (monacolin K, lovastatin) is one of
the most interesting fungat secondary metabolites. It is a competitive inhibitor of the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG CoA)
reductase (EC 1.1.1.34). This enzyme catalyses a
rate-limiting step in the biosynthesis of cholesterol, the reduction of HMG CoA to mevalonate
[1]. Mevinolin and related compounds contain a
naphthalene ring system, a/3-hydroxy lactone and
methylbutyric acid. The active form of the drug is
Correspondence to: N. Gunde-Cimerman, National Institute of
Chemistry, Hajdrihova 19, 61115 Ljubljana, Slovenia.
the corresponding/3-hydroxyacid [2]. Mevinolin is
a potent hypocholaesterolemic and hypolipaemic
agent and an effective substance for the treatment of atherosclerosis, ischaemic heart disease
and hypercholesterolaemia [3].
The first inhibitor from the series of similar
compounds (ML-236B), discovered by Endo et al.
[4] in 1976, was a metabolite of the fungus Penicillium citrinum. Later it was shown that this
compound is identical to the fungicidic compound compactin [5]. The next inhibitor, a
metabolite of the fungus Monascus ruber, named
monacolin K, was discovered by the same authors
in 1979 [6]. Shortly afterwards a secondary
metabolite, mevinolin of the soil dwelling fungus
Aspergillus terreus, was isolated [7]. Both com-
204
pounds have the same structure. After extensive
screening of soil microorganisms, it was reported
that the following fungi also synthesize ML-236B
or monacolin K: Penicillium brevicompactum,
Pythium ultimum, Hypomyces chrysospermus, Paecilomyces sp., Eupenicillium sp., Trichoderma longibrachiatum, T. pseudokoningii, Phoma sp., Doratomyces nanus and Gymnoascus umbrinus [8].
Later it was reported that also Scopulariopsis s p .
produces a H M G CoA reductase inhibitor [9].
Much attention is still paid to the discovery of
new potential producers of the inhibitor. Therefore we have decided to test fungi from our
culture collection, with special emphasis on higher
fungi, which are known to have different
metabolic abilities, especially anti-tumour activity
[10] and have a long tradition of successful use in
oriental medicine.
Materials and Methods
Organisms
380 fungal strains were from the Fungal Culture Collection (MZKIBK), at the National Institute of Chemistry, Ljubljana.
Preparation of the inoculum
The cultures were maintained on agar slants of
different composition, mostly beer wort agar and
potato dextrose agar. The slants were incubated
at 30°C for 14 days. The spore suspension was
prepared by suspending the spores from the slant
in 20 ml of water in case of moulds or by scratching the mycelium of basidiomycetous fungi and
suspending it in the same quantity of water.
Media and fermentation conditions
All fungi were cultivated in a two-step submerged fermentation. Firstly, on a seed medium
according to Alberts et al. [7]. The fungi were
cultivated 1 day on a rotary shaker at 220 rpm
and 30°C. Secondly, 10% of the seed broth was
used as inoculum for the productive substrate,of
the following composition (per 100 ml): 5.0 g
glucose, 2.0 g beer yeast, 3.0 g tomato paste, 2.0 g
oat meal, 1.0 g Na acetate, 0.5 g (NH4)2SO 4, 0.2
g KH 2PO4, 1.0 ml solution of trace elements, tap
water to 100 ml, pH 7.0.
The flasks were shaken for 5 days (for moulds)
or 10 days (for higher fungi) at 220 rpm on a
rotary shaker.
Analysis of mevinolin
The p H value of the broth samples was adjusted to 3.0 and an equal volume of methanol
was added. Extraction was done for 1 h at 200
rpm and 30°C. The samples were filtered and the
filtrates analysed. The first screening by TLC was
developed; Merck TLC aluminium sheets (Silica
gel 60 F 254) were used. The mobile phase was
dichlormethane and ethylacetate (7:3). After the
plates were developed three times they were
stained with iodine vapours. Mevinolin was determined by HPLC as described by Monaghan et al.
[11]. Mevacor (Merck) tablets were used as standard. One tablet was dissolved in a water:methanol mixture at pH 3.0.
Results and Discussion
The main goal of screening was to find new, as
yet unknown producers of the H M G CoA reductase inhibitor mevinolin.
380 fungal strains of 50 different genera and
143 species were tested. Nearly half (43%) showed
a positive result after TLC analysis, but this result
was confirmed by H P L C analysis for only 22%.
6% of all fungi tested contained only traces of
mevinolin (less than 1 mg 1-1), whereas 11%
synthesized higher amounts.
The following fungi, described by other investigators as having hypocholesterolemic activity: As-
pergillus terreus, Doratomyces nanus, Eupenicillium sp., Gymnoascus umbrinus, Hypomyces
chrysospermus, Paecylomyces sp., Phoma sp.,
Pythium ultimum, Trichoderma longibrachiatum,
T. pseudokoningii and Scopulariopsis sp. were also
tested in our screening.
Mevinolin production was detected only in
strains of Aspergillus terreus (up to 100 mg 1-1),
the best known producer of mevinolin, and also
with Paecilomyces varioti and Pythium ultimum.
The species of lower fungi, where such an activity
205
has not yet been described but where mevinolin
in the extracts was found in higher concentrations
than 1 mg 1-1 but not more than 4.5 mg 1-1 (for
Trichoderma viride) included: Aspergillus flauus,
A. niger, A. repens, A. versicolor, Penicillium variabile, Pleospora herbarum and Trichoderma viride.
More interesting results were obtained with
higher, basidiomycetous fungi that have not been
reported as producers of hypocholesterolaemic
substances. The only exception is Lentinus edodes, where the active substance eritadenine was
isolated and its structure determined [12].
Traces of mevinolin (less than 1 mg 1-~) were
found in the extracts of the following higher
fungi: Agrocybe aegerita, Trametes versicolor,
Agaricus bisporus and Volvariella colvacea.
Especially surprising were the results for the
genus Pleurotus which is not mentioned in the
literature as a producer of mevinolin. All 24
tested strains of different species showed, in
methanol extracts, at least traces of activity and
up to 41.0 mg 1-1 of mevinolin.
The first results obtained during screening
were confirmed in submerged fermentation experiments with P. sapidus, as well as in surface
fermentations with P. saca and the inhibitor was
also determined in methanol extracts of commercially available sporocarps of P. ostreatus. A
patent application has been filed using the genus
Pleurotus as a producer of hypocholesterolaemic
substances [13].
Fungi of the genus Pleurotus, especially P.
ostreatus with the common name oyster mushroom, have anti-tumour activity [14]. They are
also known for their nutritional benefit mostly
because of their high crude fibre content (8%)
and high protein content (10-30% of dry weight)
[151.
The results are presented in Table 1.
Independently, Bobek and colleagues discovered that addition of 2 - 4 % dried mushrooms to a
high-cholesterol diet given to hereditary hypercholesterolaemic rats [16] or Syrian hamsters with
hyperlipoproteinemia [17] or chronic alcohol intake [18] caused a significant reduction of serum
and liver lipids in these animals, especially regarding L D L and V L D L cholesterol, reduced up
to 80% (VLDL). Neither the chemical composi-
Table 1
Mevinolin (mg l - l ) produced by different Pleurotus strains
after 10 days of fermentation
Strain
Mevinolin (mg 1-1)
P. ostreatus
G- 9
G-18
G-20
G-17
G-14
G- 7
G-12
G-13
G-15
G-19
G-10
G- 8
G-21
G-16
G-11
0.4
0.5
0.5
0.8
1.0
1.6
1.8
1.9
2.1
2.7
3.2
5.6
15.0
23.5
27.3
P. saca
G-22
G-23
5.0
23.0
P. sapidus
G-24
41.0
Pleurotus sp.
G-25
G-29
G-30
G-27
G-26
G-28
0.7
1.7
1.7
1.8
1.9
2.2
tion of high-density lipoproteins nor the serum
concentrations were affected by the mushroom.
They attributed this effect to an isolated polysaccharide and a non-solvent residue of the ethanol
extract of the mushroom that they added separately to the diet, but it emerged that only the
whole dried sporocarp of Pleurotus ostreatus
showed such a hypocholesterolaemic activity.
Therefore they suggested that Pleurotus ostreatus
contained an unknown hypolipaemic substance.
On the basis of our results we propose that
this substance is an inhibitor of H M G CoA reductase, according to our H P L C analyses probably mevinolin. Isolation of the substance and determination of its chemical and biological activity
are presently under investigation.
206
Acknowledgements
With thanks to Darja Oven and Irena Skraba
for excellent technical assistance and to Mojca
Ben~ina for HPLC analysis.
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