INTRODUCTION
Although soil-derived microorganisms have been inten-sively
screened as a source of therapeutically important mol-ecules over a
half century1), the frequency of discoveringstructurally new
compounds is apparently decreasing theseyears. This trend seems to
imply that the easily accessiblemicroorganisms in soil had been
exhausted and there is aneed to seek unutilized microorganisms from
unexploredsources. Since the role of natural products in the drug
dis-covery is still large, new approaches such as the utilizationof
eDNA2), combinatorial biosynthesis3) and screening ofmicroorganisms
from extreme environments4) are investi-gated to discover novel
chemical structures.
It is likely that the diversity of secondary metabolites
reliesmore or less on the isolation source, namely, the habitat
ofthe producers. As for the interrelationship between plantsand
microorganisms, a couple of evidences have been pro-vided
suggesting the exchange or the transfer of biosynthet-ic gene
cluster beyond the kingdom. Taxol, an antitumorditerpene, was first
isolated from Pacific yew, Taxus brevi-folia. Recently, the
production of taxol was identified in theculture broth of a fungus
Taxomyces andreannae which is anendophyte of Pacific yew5). Another
example is the isolationof maytansine from a Celastraceae plant,
Maytenus ovatus,and ansamitocin from Actinosynnnema sp6).
Maytansine andansamitocin are comprised of an ansamycin backbone
and afatty acid side chain, and the structure of their
ansamycinbackbone is identical. These findings suggest that
endo-phytes possibly possess metabolic genes different from
themicroorganisms in other habitat.
We have identified several new bioactive compoundsfrom
actinomycetes isolated from live plants (Fig. 1). Twonew novobiocin
analogs7) were produced by Streptomycesfrom Aucuba japonica, and
cedarmycins8) by Streptomycesfrom Cryptomeria japonica as
antimicrobial metabolites.Fistupyrone9) is a metabolite of
Streptomyces from Alliumfistulosum which inhibits the infection of
Alternaria brassi-cicola to Brassica plant. Furthermore, in the
culture broth ofS. hygroscopicus from Pteridium aquilinum were
foundpteridic acids that induce the formation of adventitious
rootsin hypocotyl of kidney beans with the effectiveness
equiva-lent to that of indoleacetic acid, a plant hormone, at 1
nM10).
6-Prenylindole11) and clethramycin12) are antifungal
metabo-lites from Streptomyces sp. Anicemycin is a potent
antitu-mor antibiotic produced by S. thermoviolaceus isolated froma
leaf of Aucuba japonica. These findings indicate that plantis a
potential isolation source of strains producing new bio-active
molecules. In this article, our recent results on thescreening of
novel bioactive compounds from plant-associ-ated actinomycetes are
described.
1. Isolation of actinomycetes from live plantsIn 1978, Hasegawa
et al first reported the isolation of an
actinomycete which was neither symbiotic nor pathogenicbut
associated host-specifically with plant and the identifi-cation of
a new genus Actinosynnema13). Later, Okazaki etal investigated the
plants inhabiting in seashores and provedthe existence of
endophytic actinomycetes in leaves byshowing the scanning electron
micrographs of aerial hyphaand spore chains growing in plants14).
These studies prompt-ed us to investigate thoroughly the
distribution of actino-mycetes in herbaceous and arbor plants along
with theirpotency of producing new bioactive compounds15).
Plant samples were collected in Toyama and Miyagi pre-fectures,
Japan. In order to compare the distribution of actin-omycetes in
leaves, stems and roots of a whole plant, wechose healthy seedlings
with no apparent physical or physi-ological damages. Samples were
surface-sterilized, and in-cubated on an agar plate at 32C for a
month. The numbersof isolates were determined by counting the
colonies differ-ent from each other by macroscopic observation of
mor-phology on a Bn-2 agar slant. From 24 species of herbaceousand
arbor plants, 398 actinomycete strains were isolated.Actinomycetes
were isolated from all plants used in thisstudy, regardless of
herbaceous or arbor, or wild or agricul-tural species, suggesting
their wide distribution in associa-tion with plant in natural
environment. These strains wereused for the screening of bioactive
compounds.
2. Fistupyrone, an inhibitor of spore germination ofAlternaria
brassicicolaAlternaria brassicicola is the cause of black leaf
spot, a
major disease of cultivated Brassica plants. In the screeningof
the inhibitor of infection by A. brassicicola to Chinesecabbage, we
identified fistupyrone in the fermentation broth
63
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)
Actinomycetologica (2004) 18:6366 VOL. 18, NO. 2
*Corresponding author. Phone: +81-766-56-7500. Fax:
+81-776-56-2498. E-mail: [email protected]
of Streptomyces sp. TP-A0569 which was isolated from aleaf of
spring onion, Allium fistulosum. Although fistupyronedoes not show
the in vitro antifungal activity against A. bras-sicicola, it
completely inhibits the infection of A. brassici-cola by
pretreating the seedlings with 100 ppm of the com-pound. Further
analysis revealed that fistupyrone does notgive any effect on the
growing hyphae but specifically sup-presses the spore germination
at 0.1 ppm16). In addition, fis-tupyrone doe not show any activity
against A. alternata, apathogen of apple and pear trees.
3. Pteridic acid, an auxin-like plant growth promoterIn the
screening of plant growth regulators, pteridic acids
A and B were found in the culture broth of S.
hygroscopicusTP-A0451 isolated from a stem of bracken, Pteridium
aquil-inum. Pteridic acids A and B are stereoisomers regarding
tothe spiro carbon. Pteridic acids are probably biosynthesizedin
the biosynthetic pathway similar to that for azalomycin Bbecause
the absolute configurations of hydroxyl and methylgroups in
pteridic acid and azalomycin B are identical.Pteridic acids inhibit
the rice germination at 100 ppm, butvery surprisingly, pteridic
acid A promotes the root elonga-tion at 20 ppm. Furthermore,
pteridic acid A induces theadventitious root formation of the
kidney bean hypocotyl at1 nM as effectively as indoleacetic acid, a
native plant growthhormone. There is no report on the microbial
secondarymetabolites that show plant growth promotion at such
anextremely low concentration except for plant hormones.
4. Clethramycin, an inhibitor of pollen tube growthIn pollen
tube growth, actin/myosin cytoskeleton plays an
important role in the transport of the vesicles containing
pre-cursors for cell wall biosynthesis from the sites of their
syn-thesis to the growing pollen tube tip. This process is
inhibitedby cytochalasin or latrunculin B, an inhibitor of actin
poly-merization, and therefore pollen tube growth is also
inhib-ited. An inhibitor of cytoskeletal function is expected to
bea tool to probe the cell function and further to be a lead
fortherapeutic agents. Clethramycin is an inhibitor of pollentube
growth produced by S. hygroscopicus TP-A0326. Itshows antifungal
activity against Candida albicans andCryptococcus neoformans with
the MIC of 1 mg/ml. Al-though the relationship between pollen tube
growth and theantifungal action is obscure, the cell wall
biosynthesis is thekey event in both processes.
5. Cedarmycin, an antifungal butyrolactoneCedarmycins A and B
were isolated from the culture broth
of Streptomyces sp. TP-A0456 which was isolated from atwig of
cedar, Cryptomeria japonica. Cedarmycin is an esterof a
butyrolactone and a fatty acid. The structure reminds usof A-factor
and related microbial hormones in Streptomyces.Biosynthetically,
cedarmycin is presumably related to thesebutyrolactone hormones and
supposed to be derived from acoupling of two C-3 intermediates that
derived from the EMPpathway. Cedarmycin A shows in vitro antifungal
activityagainst Candida glabrata with the MIC of 0.4 mg/ml.
ACTINOMYCETOLOGICA VOL. 18, NO. 2
64
Fig. 1. Secondary metabolites ifolated from plant-associated
actinomycetes.
6. Other novel metabolites from plant-associated
actin-omycetes7-Demethylnovobiocin and 5-demethylnovobiocin
were isolated from Streptomyces sp. TP-A0556. This
strainproduces novobiocin as a major metabolite. Isolation yieldof
the demethyl analogs was 1~3% of that of novobiocin.Antimicrobial
activity of the demethylnovobiocins wasweaker than that of
novobiocin. The production of the mostbioactive congener,
novobiocin, is highest among the threecongeners produced by strain
TP-A0556. It is likely that theproduction of antibiotics has the
inevitability for the pro-ducer.
6-Prenylindole was isolated from the culture broth
ofStreptomyces sp. TP-A0595. This simple molecule shows
asignificant antifungal activity against plant pathogens,
A.brassicicola and Fusarium oxysporum. 6-Prenylindole wasfirst
reported as a component of the liverwort (Hepaticae).Therefore,
this is an additional example of the isolation ofthe same compound
from plant and microorganism. After theisolation of 6-prenylindole
from strain TP-A0595, we iden-tified this compound is often
produced by Streptomyces in-cluding the pteridic acid-producing
strain.
Anicemycin is a novel cytotoxic substance produced by
S.thermoviolaceus TP-A0648. It is a new analog of spicamycinand
septacidin. Anicemycin possesses an unsaturated fattyacid side
chain while the fatty acid part of spicamycin andseptacidin is
saturated. The absolute configuration of anice-mycin is not yet
determined. Anicemycin shows the cytoci-dal activity against tumor
cell lines with the IC50 of less than1 nM. Although this class of
compounds has an outstandingcytotoxicity, anicemycin is the third
example of isolation.
CONCLUDING REMARKS
What we expect natural products is their structure diver-sity
that cannot be created by chemical synthesis or rationaldesign.
Although it is not so simple to rationally assess thepotency of
natural products in drug discovery, lactacystinderivatives,
geldanamycin analogs and epothilone are thehopeful examples of
microbial secondary metabolites thatare currently investigated for
therapeutic usages. In addition,natural products have driven the
development of basicresearch over the chemistry and biology. I
believe that theimpact given by natural products will not be
changed infuture.
ACKNOWLEDGEMENTS
It was my great honor to receive the Hamada Award of theSociety
for Actinomycetes Japan (SAJ). I express my grati-tude to the award
nomination committee and the SAJ boardmembers. This research was
initiated by the inspiration ofProfessor Tamotsu Furumai (Toyama
Prefectural Univer-sity) who has continuously encouraged and
supported me. Iam most grateful to all colleagues and collaborators
whose
names are listed in References. I express special thanks
toProfessor Toshikazu Oki (Tamagawa University) who gaveme an
opportunity to engage in the research on natural prod-ucts.
REFERENCES
1) Fenical, W: Chemical studies of marine bacteria: developinga
new resource. Chem. Rev. 93: 16731683, 1993
2) Brady, S. F., C. J. Chao & J. Clardy: New natural product
fam-ilies from an environmental DNA (eDNA) gene cluster. J.
Am.Chem. Soc. 124: 99689969, 2002
3) Hutchinson, C. R: Engineering actinomycetes genes to
makeknown and new drugs. Actinomycetologica 16: 4853, 2002
4) Phoebe, Jr., C. H., J. Combie, F. G. Albert, K. V. Tran,
J.Cabrera, H. J. Correira, Y. Guo, J. Lindermuth, N. Rauert,
W.Galbraith & C. P. Selitrennikoff: Extremophilic organisms
asan unexplored source of antifungal compounds. J. Antibiot.
54:5665, 2001
5) Stierle, A., G. Strobel, D. Stierle, P. Grothaus & G.
Bignami:The search for a taxol-producing microorganism among
theendophytic fungi of the Pacific yew, Taxus brevifolia. J.
Nat.Prod. 58: 13151324, 1995
6) Higashide, E., M. Asai, K. Ootsu, S. Tanida, Y. Kozai,
T.Hasegawa, T. Kishi, Y. Sugino & M. Yoneda: Ansamitocin,
agroup of novel maytansinoid antibiotics with antitumour
prop-erties from Nocardia. Nature 270: 721722, 1977
7) Sasaki, T., Y. Igarashi, N. Saito & T. Furumai:
TPU-0031-Aand B, new antibiotics of the novobiocin group produced
byStreptomyces sp. TP-A0556. J. Antibiot. 54: 441447, 2001
8) Sasaki, T., Y. Igarashi, N. Saito & T. Furumai:
CedarmycinsA and B, new antimicrobial antibiotics from Streptomyces
sp.TP-A0456. J. Antibiot. 54: 567572, 2001
9) Igarashi, Y., M. Ogawa, Y. Sato, N. Saito, R. Yoshida,
H.Kunoh, H. Onaka & T. Furumai: Fistupyrone, a novel
inhibitorof the infection of Chinese cabbage by Alternaria
brassicico-la, from Streptomyces sp. TP-A0569. J. Antibiot. 53:
11171122, 2000
10) Igarashi, Y., T. Iida, R. Yoshida & T. Furumai: Pteridic
acidsA and B, novel plant growth promoters with auxin-like
activ-ity from Streptomyces hygroscopicus TP-A0451. J. Antibiot.55:
764767, 2002
11) Sasaki, T., Y. Igarashi, M. Ogawa & T. Furumai:
Identificationof 6-prenylindole as an antifungal metabolite of
Streptomycessp. TP-A0595 and synthesis and bioactivity of
6-substitutedindoles. J. Antibiot. 55: 10091012, 2002
12) Furumai, T., T. Yamakawa, R. Yoshida & Y.
Igarashi:Clethramycin, a new inhibitor of pollen tube growth with
anti-fungal activity from Streptomyces hygroscopicus TP-A0623.I.
Screening, taxonomy, fermentation, isolation and
biologicalproperties. J. Antibiot. 56: 700704, 2003
13) Hasegawa, T., M. P. Lechevalier & H. A. Lechevalier:
Newgenus of the Actinomycetales: Actinosynnema gen. nov. Int.J.
Syst. Bacteriol. 28: 304310, 1978
14) Okazaki, T., K. Takahashi, M. Kizuka & R. Enokita:
Studieson actinomycetes isolated from plant leaves. Annu.
Rep.Sankyo Res. Lab. 47: 97106, 1995
15) Igarashi, Y., T. Iida, T. Sasaki, N. Saito, R. Yoshida &
T.Furumai: Isolation of actinomycetes from live plants and
eval-
ACTINOMYCETOLOGICA VOL. 18, NO. 2
65
