Actinomycetologica (2004) 18:6366

VOL. 18, NO. 2

Award Lecture
Screening of Novel Bioactive Compounds
from Plant-Associated Actinomycetes
Yasuhiro Igarashi*
Biotechnology Research Center, Toyama Prefectural University, 5180 Kurokawa, Kosugi,
Toyama 939-0398, Japan
(Received Sep. 30, 2004)

6-Prenylindole11) and clethramycin12) are antifungal metabolites from Streptomyces sp. Anicemycin is a potent antitumor antibiotic produced by S. thermoviolaceus isolated from
a leaf of Aucuba japonica. These findings indicate that plant
is a potential isolation source of strains producing new bioactive molecules. In this article, our recent results on the
screening of novel bioactive compounds from plant-associated actinomycetes are described.

INTRODUCTION
Although soil-derived microorganisms have been intensively screened as a source of therapeutically important molecules over a half century1), the frequency of discovering
structurally new compounds is apparently decreasing these
years. This trend seems to imply that the easily accessible
microorganisms in soil had been exhausted and there is a
need to seek unutilized microorganisms from unexplored
sources. Since the role of natural products in the drug discovery is still large, new approaches such as the utilization
of eDNA2), combinatorial biosynthesis3) and screening of
microorganisms from extreme environments4) are investigated to discover novel chemical structures.
It is likely that the diversity of secondary metabolites relies
more or less on the isolation source, namely, the habitat of
the producers. As for the interrelationship between plants
and microorganisms, a couple of evidences have been provided suggesting the exchange or the transfer of biosynthetic gene cluster beyond the kingdom. Taxol, an antitumor
diterpene, was first isolated from Pacific yew, Taxus brevifolia. Recently, the production of taxol was identified in the
culture broth of a fungus Taxomyces andreannae which is an
endophyte of Pacific yew5). Another example is the isolation
of maytansine from a Celastraceae plant, Maytenus ovatus,
and ansamitocin from Actinosynnnema sp6). Maytansine and
ansamitocin are comprised of an ansamycin backbone and a
fatty acid side chain, and the structure of their ansamycin
backbone is identical. These findings suggest that endophytes possibly possess metabolic genes different from the
microorganisms in other habitat.
We have identified several new bioactive compounds
from actinomycetes isolated from live plants (Fig. 1). Two
new novobiocin analogs7) were produced by Streptomyces
from Aucuba japonica, and cedarmycins8) by Streptomyces
from Cryptomeria japonica as antimicrobial metabolites.
Fistupyrone9) is a metabolite of Streptomyces from Allium
fistulosum which inhibits the infection of Alternaria brassicicola to Brassica plant. Furthermore, in the culture broth of
S. hygroscopicus from Pteridium aquilinum were found
pteridic acids that induce the formation of adventitious roots
in hypocotyl of kidney beans with the effectiveness equivalent to that of indoleacetic acid, a plant hormone, at 1 nM10).

1. Isolation of actinomycetes from live plants

In 1978, Hasegawa et al first reported the isolation of an
actinomycete which was neither symbiotic nor pathogenic
but associated host-specifically with plant and the identification of a new genus Actinosynnema13). Later, Okazaki et
al investigated the plants inhabiting in seashores and proved
the existence of endophytic actinomycetes in leaves by
showing the scanning electron micrographs of aerial hypha
and spore chains growing in plants14). These studies prompted us to investigate thoroughly the distribution of actinomycetes in herbaceous and arbor plants along with their
potency of producing new bioactive compounds15).
Plant samples were collected in Toyama and Miyagi prefectures, Japan. In order to compare the distribution of actinomycetes in leaves, stems and roots of a whole plant, we
chose healthy seedlings with no apparent physical or physiological damages. Samples were surface-sterilized, and incubated on an agar plate at 32C for a month. The numbers
of isolates were determined by counting the colonies different from each other by macroscopic observation of morphology on a Bn-2 agar slant. From 24 species of herbaceous
and arbor plants, 398 actinomycete strains were isolated.
Actinomycetes were isolated from all plants used in this
study, regardless of herbaceous or arbor, or wild or agricultural species, suggesting their wide distribution in association with plant in natural environment. These strains were
used for the screening of bioactive compounds.
2. Fistupyrone, an inhibitor of spore germination of
Alternaria brassicicola
Alternaria brassicicola is the cause of black leaf spot, a
major disease of cultivated Brassica plants. In the screening
of the inhibitor of infection by A. brassicicola to Chinese
cabbage, we identified fistupyrone in the fermentation broth

*Corresponding author. Phone: +81-766-56-7500. Fax: +81-776-56-2498. E-mail: yas@pu-toyama.ac.jp

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Fig. 1.

VOL. 18, NO. 2

Secondary metabolites ifolated from plant-associated actinomycetes.

of Streptomyces sp. TP-A0569 which was isolated from a

leaf of spring onion, Allium fistulosum. Although fistupyrone
does not show the in vitro antifungal activity against A. brassicicola, it completely inhibits the infection of A. brassicicola by pretreating the seedlings with 100 ppm of the compound. Further analysis revealed that fistupyrone does not
give any effect on the growing hyphae but specifically suppresses the spore germination at 0.1 ppm16). In addition, fistupyrone doe not show any activity against A. alternata, a
pathogen of apple and pear trees.

4. Clethramycin, an inhibitor of pollen tube growth

In pollen tube growth, actin/myosin cytoskeleton plays an
important role in the transport of the vesicles containing precursors for cell wall biosynthesis from the sites of their synthesis to the growing pollen tube tip. This process is inhibited
by cytochalasin or latrunculin B, an inhibitor of actin polymerization, and therefore pollen tube growth is also inhibited. An inhibitor of cytoskeletal function is expected to be
a tool to probe the cell function and further to be a lead for
therapeutic agents. Clethramycin is an inhibitor of pollen
tube growth produced by S. hygroscopicus TP-A0326. It
shows antifungal activity against Candida albicans and
Cryptococcus neoformans with the MIC of 1 mg/ml. Although the relationship between pollen tube growth and the
antifungal action is obscure, the cell wall biosynthesis is the
key event in both processes.

3. Pteridic acid, an auxin-like plant growth promoter

In the screening of plant growth regulators, pteridic acids
A and B were found in the culture broth of S. hygroscopicus
TP-A0451 isolated from a stem of bracken, Pteridium aquilinum. Pteridic acids A and B are stereoisomers regarding to
the spiro carbon. Pteridic acids are probably biosynthesized
in the biosynthetic pathway similar to that for azalomycin B
because the absolute configurations of hydroxyl and methyl
groups in pteridic acid and azalomycin B are identical.
Pteridic acids inhibit the rice germination at 100 ppm, but
very surprisingly, pteridic acid A promotes the root elongation at 20 ppm. Furthermore, pteridic acid A induces the
adventitious root formation of the kidney bean hypocotyl at
1 nM as effectively as indoleacetic acid, a native plant growth
hormone. There is no report on the microbial secondary
metabolites that show plant growth promotion at such an
extremely low concentration except for plant hormones.

5. Cedarmycin, an antifungal butyrolactone

Cedarmycins A and B were isolated from the culture broth
of Streptomyces sp. TP-A0456 which was isolated from a
twig of cedar, Cryptomeria japonica. Cedarmycin is an ester
of a butyrolactone and a fatty acid. The structure reminds us
of A-factor and related microbial hormones in Streptomyces.
Biosynthetically, cedarmycin is presumably related to these
butyrolactone hormones and supposed to be derived from a
coupling of two C-3 intermediates that derived from the EMP
pathway. Cedarmycin A shows in vitro antifungal activity
against Candida glabrata with the MIC of 0.4 mg/ml.
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names are listed in References. I express special thanks to

Professor Toshikazu Oki (Tamagawa University) who gave
me an opportunity to engage in the research on natural products.

6. Other novel metabolites from plant-associated actinomycetes

7-Demethylnovobiocin and 5-demethylnovobiocin
were isolated from Streptomyces sp. TP-A0556. This strain
produces novobiocin as a major metabolite. Isolation yield
of the demethyl analogs was 1~3% of that of novobiocin.
Antimicrobial activity of the demethylnovobiocins was
weaker than that of novobiocin. The production of the most
bioactive congener, novobiocin, is highest among the three
congeners produced by strain TP-A0556. It is likely that the
production of antibiotics has the inevitability for the producer.
6-Prenylindole was isolated from the culture broth of
Streptomyces sp. TP-A0595. This simple molecule shows a
significant antifungal activity against plant pathogens, A.
brassicicola and Fusarium oxysporum. 6-Prenylindole was
first reported as a component of the liverwort (Hepaticae).
Therefore, this is an additional example of the isolation of
the same compound from plant and microorganism. After the
isolation of 6-prenylindole from strain TP-A0595, we identified this compound is often produced by Streptomyces including the pteridic acid-producing strain.
Anicemycin is a novel cytotoxic substance produced by S.
thermoviolaceus TP-A0648. It is a new analog of spicamycin
and septacidin. Anicemycin possesses an unsaturated fatty
acid side chain while the fatty acid part of spicamycin and
septacidin is saturated. The absolute configuration of anicemycin is not yet determined. Anicemycin shows the cytocidal activity against tumor cell lines with the IC50 of less than
1 nM. Although this class of compounds has an outstanding
cytotoxicity, anicemycin is the third example of isolation.

REFERENCES
1)
2)

3)
4)

5)

6)

7)

8)

9)

CONCLUDING REMARKS
What we expect natural products is their structure diversity that cannot be created by chemical synthesis or rational
design. Although it is not so simple to rationally assess the
potency of natural products in drug discovery, lactacystin
derivatives, geldanamycin analogs and epothilone are the
hopeful examples of microbial secondary metabolites that
are currently investigated for therapeutic usages. In addition,
natural products have driven the development of basic
research over the chemistry and biology. I believe that the
impact given by natural products will not be changed in
future.

10)

11)

12)

ACKNOWLEDGEMENTS
13)

It was my great honor to receive the Hamada Award of the

Society for Actinomycetes Japan (SAJ). I express my gratitude to the award nomination committee and the SAJ board
members. This research was initiated by the inspiration of
Professor Tamotsu Furumai (Toyama Prefectural University) who has continuously encouraged and supported me. I
am most grateful to all colleagues and collaborators whose

14)

15)

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