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Biotechnological Research on
Endophytic Microbes
from a tea Plant and the Parasitic Plant
Partomuan Simanjuntak
Research Center for Green Science
Fukuyama University
Research Centre for Biotehnology,Indonesian Institute of Sciences (LIPI)
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Relationship between plants and microorganisms
Plant
(eukaryota) Microorganism
Epiphyte(living outside
of a plant)
Endophyte(living inside
of a plant)
Bacterium(prokaryota)
Fungus(eukaryota)
symbiosisor
parasite
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Endophytes have an ability to bio-synthesizethe chemical constituents of the symbioticplant
Endophytes have an ability to bio-transform
the chemical constituents of the symbiotic
plant
Hypothesis - 1
Hypothesis - 2
Endophytes have an ability to bio-synthesizethe chemical constituents of the symbioticplant
Endophytes have an ability to bio-transform
the chemical constituents of the symbiotic
plant
Hypothesis - 1
Hypothesis - 2
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Collected at the Puncak area, West Java, Indonesia
The tea plant Camellia sinensis (L.) O.K.
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1) washed with water
2) treated with 75% aq. EtOH for 1.0 min3) treated with 5.3% aq. sodium
hypochlorite for 0.5 min4) treated with 75% aq. EtOH for 0.5 min5) cut using sterilized cutter
put on CMMA mediumcontaining chloramphenicol
(0.05 mg/ml)
separation of colonies
inoculation ontoPDA medium
the young stem 6 filamentous fungi
separationof colonies
Separation of the endophytic fungi from a tea plant
tea plant
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fungus family name genus name
A
B
C
D
E
F
Nectriaceae
Trichocomaceae
Schizophyllaceae
Schizophyllaceae
Valsaceae
Valsaceae
Fusarium
Penicillium
Schizophyllum
Schizophyllum
Diaporthe
Diaporthe
AB245442
AB245443
AB245444
AB245445
AB245446
AB245447
accession no.*
* DDBJ
Endophytic filamentous fungi from a tea plant were classifiedinto six species by RAPD and rDNA sequences analysis
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endophytic fungusOHO
OH
OH
OH
OH
tea plant
bio-transformedproducts ?
Endophytes have an ability to bio-transformthe chemical constituents ofCamellia sinensis
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Catechin in the tea plant Camellia sinensis
OHO
OHOH
OHOH
OHO
OHO
OHOH
galloyl
OHO
OHO
OHOH
OH
galloyl
OHO
OHOH
OHOH
R
(+)-catechin (1, CA) (-)-epicatechin R = H (2, EC)
(-)-epigallocatechin 3-O-gallate (5, EGCG)(-)-epicatechin 3-O-gallate (4, ECG)
(-)-epigallocatechin R = OH (3, EGC)
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Endophytic Diaporthe sp. (fungus E)
a closely related species toDiaporthe phaseolorum var. sojae
Screening test of an ability to bio-transform (+)-catechinby 6 endophytic fungi (fungus A ~ F)
Mediums used for screening tests
1. MY202. Nutrient broth with 0.5% NaCl3. Tryptic-glucose
4. Potato dextrose broth (PDB)5. Glucose-yeast extract-peptone
Endophytic Diaporthe sp. (fungus E)showed a bio-transformation capability against
(+)-catechin in medium 5
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Bio-transformation of (+)-catechin by Diaporthe sp. (fungus E)
cultivation for 5 dayswith shaking at 27
oC
200 ml ofglucose-yeastextract-peptone
Diaporthe sp.(fungus E)
EtOAc-solubleportion (45 mg)
2) extraction with EtOAc
inoculation
(+)-catechin(20 mg) in
MeOH
1) cultivation for one day
1) SiO2 column(CHCl3:MeOH:H2O=65:35:10,
lower phase)
2) HPLC (Capcell Pak PhenylSG-120, 250 x 4.6 mm, H2O)
biotransformedproduct
(9.5 mg, 45%)
recovered (+)-catechin(1.7 mg, 8.5%)
addition
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HPLC conditions: CAPCELL PAK Phenyl SG-120 250 x 4.6 mm, rt, 20 mM KH2PO4-CH3CN
(gradient: 0 min, 5% CH3CN 40 min, 15% CH3CN), 210 nm
0.00 10.00 20.00 30.00
0.00
500.00
time (min)
(+)-catechin (1)
biotransformed product (6)
0.00
absorbance
HPLC chromatogram of biotransformed product from (+)-catechinby Diaporthe sp. (fungus E)
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1H NMR (d6-acetone)
OHO
OHOH
OH OH
OH
R
S
The bio-transformed product from (+)-catechin
(+)-(2R,3S,4S)-3,4,5,7,3',4'-
hexahydroxyflavan(6)*
H-2: 4.84 (d, J= 9.5 Hz)H-3: 3.88 (dd, J= 3.7, 9.5 Hz)H-4: 4.47 (d, J= 3.7 Hz)
23
4
* Kristiansen K.N., Carlsberg Res. Commun., 49, 503-524 (1984).Kristiansen K.N., Carlsberg Res. Commun., 51, 51-60 (1986).
C15H14O7 (HR-FABMS)
[]D +5.5o (EtOH)
IR (KBr): 3300, 1612 cm-1
UV (EtOH): 280 nm (= 4.000)
OHO
OHOH
OHOH
(+)-catechin (1, CA)
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Bio-transformation of (-)-epicatechin by Diaporthe sp. (fungus E)
Cultivation conditions:
1. cultivation in gluc.-yeast ext.-peptone medium2. shaking (90 rpm); at 27oC for 5 days
(-)-epicatechin (2)(-)-(2R,3R,4R)-3,4,5,7,3',4'-hexahydroxyflavan (7, new)
O
OH
HO
OH
OH
OH
OHO
HO R R
R
R
2
4
nOe
Diaporthe sp.(fungus E)
39%
OH
OH
H
H
H
OHHO
. 1H NMR (d6-acetone)H-2:5.00 (br s)H-3:3.88 (dd, J=1.2, 2.2 Hz)H-4:4.75 (d, J=2.2 Hz)
7: C15H14O7 (HR-FABMS)[]D -8.4
o (EtOH)IR (KBr): 3250, 1628 cm-1
UV (EtOH): 279 nm (= 4,200)
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(-)-epicatechin (2)
O
OH
HO
OH
OH
OH
R
R
O
OH
HO
OH
OH
OH
R
R
OH
(-)-(2R,3R,4R)-3,4,5,7,3',4'-hexahydroxyflavan (7, new)
Diaporthe sp.(fungus E)
39%
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HPLC conditions: CAPCELL PAK Phenyl SG-120 250 x 4.6 mm, rt, 20 mM KH 2PO4-CH3CN(gradient: 0 min, 5% CH3CN 40 min, 15% CH3CN), 210 nm
time (min)
0.00 10.00 20.00 30.00
0.00
500.00
(-)-epicatechin (2, 2.4%)
biotransformed product (7, 39%)
0.00
absorb
ance
HPLC chromatogram of biotransformed product from (-)-epicatechinby Diaporthe sp. (fungus E)
OHO
OH
OH
OHOH
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O
OH
HO
OHOH
O-galloyl
O
OH
HO
OHOH
OH
O
OH
HO
OHOH
OH
Diaporthe sp
(fungus E)
OH
R
R
R
R
R
R
R
234
(-)-epicatechin 3-O-gallate (4) (-)-epicatechin (2) (-)-(2R,3R,4R)-3,4,5,7,3',4'-hexahydroxyflavan (7, 53%)
O
OH
HO
OHOH
O-galloyl
RR
OH Diaporthe sp
(fungus E)
O
OH
HO
OHOH
OH
RR
OH O
OH
HO
OHOH
OH
RR
R
234
OH
(-)-epigallocatechin 3-O-gallate (5) (-)-epigallocatechin (3) (-)-(2R,3R,4R)-3,4,5,7,3',4',5'-heptahydroxyflavan (8, 43%, new)
OH
Bio-transformation of ECG and EGCG by Diaporthe sp. (fungus E)
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0.00 10.0020.00 40.00
0.00
200.00
time (min)
biotransformed product (7, 53%)
(-)-epicatechin (2, 13%)
30.00
(-)-ECG (4)
absorb
ance
HPLC conditions: CAPCELL PAK Phenyl SG-120 250 x 4.6 mm, rt, 20 mM KH2PO4-CH3CN(gradient: 0 min, 5% CH3CN 40 min, 15% CH3CN), 210 nm
HPLC chromatogram of biotransformed product from (-)-ECGby Diaporthe sp. (fungus E)
OHO
OH
O
OHOH
galloyl
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0.00
time (min)
10.0020.00
200.00
40.00
0.00
(-)-epigallocatechin (3, 19%)
biotransformed product (8, 43%)
30.00
(-)-EGCG (5)
absorban
ce
HPLC conditions: CAPCELL PAK Phenyl SG-120 250 x 4.6 mm, rt, 20 mM KH2PO4-CH3CN(gradient: 0 min, 5% CH3CN 40 min, 15% CH3CN), 210 nm
HPLC chromatogram of biotransformed product from (-)-EGCGby Diaporthe sp. (fungus E)
OHO
OHO
OHOH
galloyl
OH
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Stereoselectivity
OHO
OH
OH
OH
OH
OH
S
OHO
OH
OH
OH
OH
S
R
OHO
OH
OH
OH
OH
R2OHO
OH
R2
OH
OR1
OH
R
S
OHR
Diaporthe sp.(fungus E)
3 34
3S : (+)-CA (1)
3
Insertion of4S-hydroxyl group
3R : (-)-EC (2); (-)-ECG (4)(-)-EGCG (5)
3
4
Diaporthe sp.
(fungus E)4 4
Insertion of4R-hydroxyl group
[(-)-(2R,3R,4R)-3,4,5,7,3',4'-hexahydroxyflavans (7 and 8)]
[(+)-(2R,3S,4S)-3,4,5,7,3',4'-hexahydroxyflavan (6)]
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Substrate specificity
ROHO
OH
R2
OH
OR1
OH
SOHO
OH
OH
OH
OH
OHO
OH
OH
OH
O
OH
galloyl
S
2R
2S
Diaporthe sp.(fungus E)
2
4
(+)-CA (1), (-)-EC (2),(-)-ECG (4), (-)-EGCG (5)
Diaporthe sp.(fungus E)
2
(-)-CA (10), (+)-EC (11)
recovered
3,4-cis-dihydroxyflavan (6,7,8)
(-)-GCG (9) (-)-GC (17)
hydrolysis
2
oxidation at C-4 ROHO
OH
R2
OH
OH
OH
2
OH
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Endophytes have an ability to bio-synthesizethe chemical constituents of the symbioticplant
Endophytes have an ability to bio-transformthe chemical constituents of the symbiotic
plant
Hypothesis - 1
Hypothesis - 2
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Collected at the Puncak area, West Java, Indonesia
The Parasitic plant Dendrophthoe pentandraof the tea plant Camellia sinensis
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(+)-catechin from the parasitic plantDendrophthoe pentandra of a tea plant
Dendrophthoe pentandra(Stem and leaves)
70% aq.acetone extract
partitioned into EtOAc and water
SiO2 column chr.
EtOAc-soluble portion H2O soluble portion
Fr. 1 Fr. 2 Fr. 3 Fr. 4
1) extracted with 70% aq.acetone2) evap. in vacuo
HPLC(+)-catechinIdentified by []D, UV, IR and NMR
OHO
OH
OH
OH
OH
(+)-catechin
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Separation
Screening test of an ability to bio-produce (+)-catechin
parasitic plantDendrophthoe pentandra
tea plantCamellia sinensis
2 fungi(G and H)
6 fungi(A ~ F)
Mediums used for screening tests
1. Phoma medium2. S7 medium3. Glucose-peptone medium4. PDB
Endophytic fungus H showed a capabilityto bio-produce (+)-catechin in medium PDB
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in potato dextrose brothwith shaking at 28 oC,
for 3 days
Identified by[]D, UV, IR, NMR
(+)-catechin
Bio-production of (+)-catechin by endophytic fungus H
Bio-products
Fungus H,
a new fungus, Phomopsis sp.
0.00
0.02
0.04
0.06
0 5 10 15 20 25 30 35 40 45(min)
0.05
0.03
0.01
CAPCELL PAK C18, 20 mM Phosphate buffer-acetonitrile (gradient), 210 nm
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OHO
OH
OH
OH
OH
(+)-catechin
Parasitic plantDendrophthoe pentandra
Tea plantCamellia sinensis (L.) O.K.
EndophytePhomopsis sp.production
Production ??separation
What`s the genuine producer of (+)-catechin ?
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Conclusion
1. The endophytic fungus Diaporthe sp. from the tea plant
Camellia sinensis has an ability to bio-transform the tea
catechin to the corresponding 3,4-cis-dihydroxyflavan
derivatives
2. The endophytic fungus Phomopsis sp. from the parasitic
plant Dendrophthoe pentandra has an ability to bio-
synthesize (+)-catechin
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Law of Applied Microbiology- The microorganism is always right,
your friend and sensitive partner
- They are no stupid microorganism
- Microorganisms can (will) do anything
- Microorganism are smarter, wiser, more
energetic than chemist, microbiologist, etc
- If you take of your (microbial) friends, they
will take care of your future
(and you will live happily everyafter)
Law of Applied Microbiology- The microorganism is always right,
your friend and sensitive partner
- They are no stupid microorganism
- Microorganisms can (will) do anything
- Microorganism are smarter, wiser, more
energetic than chemist, microbiologist, etc
- If you take of your (microbial) friends, they
will take care of your future
(and you will live happily everyafter)
(D. Parlman, 1979)
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The Puncak area, West Java, Indonesia
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Collaboration between Fukuyama Universityand
Indonesian Institute of Sciences
Fukuyama University
Dr. Made Sri Prana
Dr. Irawati
Dr. Andria Agusta
Dr. Hirotaka Shibuya
Dr. Kazuyoshi Ohashi
Mr. Shoji Maehara
Ms. Chinami KitamuraMr. Masakazu Tokuhisa
Indonesian Institute
of Sciences
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between plant andendophytic microbes
Microbes which associates with living tissue of the
plant. The type of interaction between endophyte and its
host plant usually mutualism symbiosis. The plant
providing nutrients for microbe, while the microbe
transform and produce bioactive compounds
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O
OH
OH
H3CO OR
HOHO
COOH
COOH
COOH
COOH
O
R = H, benzophenoneR = glucose, benzophenone Cycloargentenol -sitosterol
asam palmitat
asam oleat
asam linoleat
asam linolenat
O
O
OH
OH
HOH2C
OH
CH2OH
OH
HO
Sucrose
(-D-glucopyranosyl--D-fructofuranoside)
CH2OH
O
OH
HO
HO
HOH2C
OH
-D-glucopyranose
Chemical compounds of mahkota dewa,
Phaleria macrocarpa
Bioactive compound
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Produksi mikroba endofit C. aeruginosa (Jepang)
Produksi mikroba endofit C. domestica/C.longa (Indonesia)
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