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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)
Relationship between plants and microorganisms

Plant symbiosis
(eukaryota) or Microorganism
parasite

Endophyte
(living inside
of a plant)

Epiphyte
(living outside
of a plant)
Fungus Bacterium
(eukaryota) (prokaryota)
Hypothesis - 1

Endophytes have an ability to bio-transform


the chemical constituents of the symbiotic
plant

Hypothesis - 2

Endophytes have an ability to bio-synthesize


the chemical constituents of the symbiotic
plant
The tea plant Camellia sinensis (L.) O.K.

Collected at the Puncak area, West Java, Indonesia


Separation of the endophytic fungi from a tea plant

the young stem tea plant 6 filamentous fungi


1) washed with water
2) treated with 75% aq. EtOH for 1.0 min
3) treated with 5.3% aq. sodium separation
hypochlorite for 0.5 min of colonies
4) treated with 75% aq. EtOH for 0.5 min
5) cut using sterilized cutter

separation of colonies

put on CMMA medium inoculation onto


containing chloramphenicol PDA medium
(0.05 mg/ml)
Endophytic filamentous fungi from a tea plant were classified
into six species by RAPD and rDNA sequences analysis

fungus family name genus name accession no.*

A Nectriaceae Fusarium AB245442

B Trichocomaceae Penicillium AB245443

C Schizophyllaceae Schizophyllum AB245444

D Schizophyllaceae Schizophyllum AB245445

E Valsaceae Diaporthe AB245446

F Valsaceae Diaporthe AB245447

* DDBJ
Endophytes have an ability to bio-transform
the chemical constituents of Camellia sinensis

OH
OH

HO O
endophytic fungus
OH
OH

tea plant

bio-transformed
products ?
Catechin in the tea plant Camellia sinensis

OH OH
OH OH

HO O HO O
R

OH OH
OH OH

(+)-catechin (1, CA) (-)-epicatechin R = H (2, EC)


(-)-epigallocatechin R = OH (3, EGC)

OH OH
OH OH

HO O HO O
OH

O galloyl O galloyl
OH OH

(-)-epicatechin 3-O-gallate (4, ECG) (-)-epigallocatechin 3-O-gallate (5, EGCG)


Screening test of an ability to bio-transform (+)-catechin
by 6 endophytic fungi (fungus A ~ F)

Mediums used for screening tests


1. MY20 4. Potato dextrose broth (PDB)
2. Nutrient broth with 0.5% NaCl 5. Glucose-yeast extract-peptone
3. Tryptic-glucose

Endophytic Diaporthe sp. (fungus E)


showed a bio-transformation capability against
(+)-catechin in medium 5

Endophytic Diaporthe sp. (fungus E)

a closely related species to


Diaporthe phaseolorum var. sojae
Bio-transformation of (+)-catechin by Diaporthe sp. (fungus E)

addition
inoculation cultivation for 5 days
with shaking at 27oC
(+)-catechin
200 ml of (20 mg) in
Diaporthe sp. glucose-yeast MeOH
(fungus E) extract-peptone

1) cultivation for one day


2) extraction with EtOAc
biotransformed
product
(9.5 mg, 45%) 1) SiO2 column
(CHCl3:MeOH:H2O=65:35:10,
lower phase) EtOAc-soluble
portion (45 mg)
2) HPLC (Capcell Pak Phenyl
recovered (+)-catechin SG-120, 250 x 4.6 mm, H2O)
(1.7 mg, 8.5%)
HPLC chromatogram of biotransformed product from (+)-catechin
by Diaporthe sp. (fungus E)

biotransformed product (6)


absorbance

500.00

(+)-catechin (1)

0.00
0.00 10.00 20.00 30.00 40.00

time (min)

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
The bio-transformed product from (+)-catechin

OH
C15H14O7 (HR-FABMS)
OH
[ƒ¿]D +5.5o (EtOH)
HO O R IR (KBr): 3300, 1612 cm-1
2

4
3 S UV (EtOH): 280 nm (= 4.000)
OH 1
H NMR (d6-acetone)
OH OH
H-2: ƒÂ4.84 (d, J = 9.5 Hz)
H-3: ƒÂ3.88 (dd, J = 3.7, 9.5 Hz)
(+)-(2R,3S,4S)-3,4,5,7,3',4'-
H-4: ƒÂ4.47 (d, J = 3.7 Hz)
hexahydroxyflavan (6)*

OH
OH * Kristiansen K.N., Carlsberg Res. Commun., 49, 503-524 (1984).
Kristiansen K.N., Carlsberg Res. Commun., 51, 51-60 (1986).
HO O

OH
OH

(+)-catechin (1, CA)


Bio-transformation of (-)-epicatechin by Diaporthe sp. (fungus E)

Cultivation conditions:
1. cultivation in gluc.-yeast ext.-peptone medium
2. shaking (90 rpm); at 27oC for 5 days

OH
OH nOe
OH
OH
HO H
Diaporthe sp. O
HO O R (fungus E) H 2
4 H
R 39% R
HO R
OH HO OH
OH (-)-(2R,3R,4R)-3,4,5,7,3',4'-
(-)-epicatechin (2) hexahydroxyflavan (7, new)

7: C15H14O7 (HR-FABMS) . 1
o
H NMR (d6-acetone)
[α]D -8.4 (EtOH) H-2: δ5.00 (br s)
IR (KBr): 3250, 1628 cm-1 H-3: δ3.88 (dd, J=1.2, 2.2 Hz)
UV (EtOH): 279 nm ( = 4,200) H-4: δ4.75 (d, J=2.2 Hz)
OH OH
OH OH
Diaporthe sp.
HO O R (fungus E) HO O R

R 39% R
OH OH
OH OH OH
(-)-epicatechin (2) (-)-(2R,3R,4R)-3,4,5,7,3',4'-
hexahydroxyflavan (7, new)
HPLC chromatogram of biotransformed product from (-)-epicatechin
by Diaporthe sp. (fungus E)

OH
500.00 OH
biotransformed product (7, 39%)
HO O
absorbance

OH
OH

(-)-epicatechin (2, 2.4%)

0.00

0.00 10.00 20.00 30.00 40.00


time (min)

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
Bio-transformation of ECG and EGCG by Diaporthe sp. (fungus E)

OH OH OH
OH OH OH
HO OR Diaporthe sp HO OR HO OR
R R 2
R
(fungus E) 43
O-galloyl OH R OH
OH OH OH OH
(-)-epicatechin 3-O-gallate (4) (-)-epicatechin (2) (-)-(2R,3R,4R)-3,4,5,7,3',4'-
hexahydroxyflavan (7, 53%)

OH OH OH
OH OH OH
HO OR HO OR HO OR
OH Diaporthe sp OH 2 OH
R R 4 3R
O-galloyl (fungus E) OH R OH
OH OH OH OH
(-)-epigallocatechin 3-O-gallate (5) (-)-epigallocatechin (3) (-)-(2R,3R,4R)-3,4,5,7,3',4',5'-
heptahydroxyflavan (8, 43%, new)
HPLC chromatogram of biotransformed product from (-)-ECG
by Diaporthe sp. (fungus E)

OH
OH
200.00
HO O
biotransformed product (7, 53%)
absorbance

O galloyl
OH
(-)-ECG (4)
(-)-epicatechin (2, 13%)

0.00

0.00 10.00 20.00 30.00 40.00


time (min)

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 (-)-EGCG
by Diaporthe sp. (fungus E)

OH
OH

200.00 HO O
biotransformed product (8, 43%) OH
absorbance

O galloyl
OH
(-)-epigallocatechin (3, 19%) (-)-EGCG (5)

0.00

0.00 10.00 20.00 30.00 40.00

time (min)

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
Stereoselectivity
OH OH
OH OH

HO O Diaporthe sp. HO O
(fungus E)
3 S 3S
4 4
OH S OH
OH OH OH

3S : (+)-CA (1) Insertion of 4S-hydroxyl group


[(+)-(2R,3S,4S)-3,4,5,7,3',4'-
hexahydroxyflavan (6)]

OH OH
OH OH
HO O Diaporthe sp. HO O
R2 (fungus E) R2
3 R 3 R
4 4
OR1 R OH
OH OH OH
3R : (-)-EC (2); (-)-ECG (4) Insertion of 4R-hydroxyl group
(-)-EGCG (5) [(-)-(2R,3R,4R)-3,4,5,7,3',4'-
hexahydroxyflavans (7 and 8)]
Substrate specificity

2R OH OH
OH OH
HO OR HO OR
2
R2 oxidation at C-4 R2
2
OR1 Diaporthe sp. 4
OH
OH (fungus E)
OH OH
(+)-CA (1), (-)-EC (2), 3,4-cis-dihydroxyflavan (6,7,8)
(-)-ECG (4), (-)-EGCG (5)

2S OH OH
OH OH
recovered
HO OS HO OS
2
OH 2

O galloyl OH Diaporthe sp.


OH OH (fungus E)
hydrolysis
(-)-GCG (9) (-)-GC (17) (-)-CA (10), (+)-EC (11)
Hypothesis - 1

Endophytes have an ability to bio-transform


the chemical constituents of the symbiotic
plant

Hypothesis - 2

Endophytes have an ability to bio-synthesize


the chemical constituents of the symbiotic
plant
The Parasitic plant Dendrophthoe pentandra
of the tea plant Camellia sinensis

Collected at the Puncak area, West Java, Indonesia


(+)-catechin from the parasitic plant
Dendrophthoe pentandra of a tea plant

Dendrophthoe pentandra
OH
(Stem and leaves)
OH

O
1) extracted with 70% aq.acetone
HO
2) evap. in vacuo
OH
OH 70% aq.acetone extract
(+)-catechin partitioned into EtOAc and water

EtOAc-soluble portion H2O soluble portion


SiO2 column chr.

Fr. 1 Fr. 2 Fr. 3 Fr. 4


HPLC
Identified by []D, UV, IR and NMR (+)-catechin
Screening test of an ability to bio-produce (+)-catechin

parasitic plant tea plant


Dendrophthoe pentandra Camellia sinensis
Separation
Mediums used for screening tests
1. Phoma medium
2 fungi 2. S7 medium 6 fungi
(G and H) 3. Glucose-peptone medium (A ~ F)
4. PDB

Endophytic fungus H showed a capability


to bio-produce (+)-catechin in medium PDB
Bio-production of (+)-catechin by endophytic fungus H

in potato dextrose broth


with shaking at 28 oC,
for 3 days
Bio-products
Fungus H,
a new fungus, Phomopsis sp.
0.06

0.05
Identified by
0.04 []D, UV, IR, NMR
0.03 (+)-catechin

0.02
0.01

0.00

0 5 10 15 20 25 30 35 40 45 (min)
CAPCELL PAK C18, 20 mM Phosphate buffer-acetonitrile (gradient), 210 nm
What`s the genuine producer of (+)-catechin ?

Tea plant Parasitic plant


Camellia sinensis (L.) O.K. Dendrophthoe pentandra

Production ??
separation
OH
OH

HO O

OH Endophyte
OH production Phomopsis sp.
(+)-catechin
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
Law
LawofofApplied
AppliedMicrobiology
Microbiology
- The
- Themicroorganism
microorganism isis
always
always
right,
right,
your
yourfriend
friend
and
and
sensitive
sensitive
partner
partner

- They
- They
are
are
nono
stupid
stupid
microorganism
microorganism

- Microorganisms
- Microorganisms
can
can
(will)
(will)
dodo
anything
anything

- Microorganism
- Microorganismare
are
smarter,
smarter,
wiser,
wiser,
more
more
energetic
energetic
than
than
chemist,
chemist,
microbiologist,
microbiologist,etc
etc

- If- If
youyou
take
take
ofof
your
your (microbial)
(microbial) friends,
friends,
they
they
will
will take
takecare
care
ofofyour
your
future
future
(and
(and
you
youwill
willlive
live
happily
happily everyafter)
everyafter)
(D. Parlman, 1979)
The Puncak area, West Java, Indonesia
Collaboration between Fukuyama University
and
Indonesian Institute of Sciences

Fukuyama University Indonesian Institute


of Sciences
Dr. Hirotaka Shibuya Dr. Made Sri Prana
Dr. Kazuyoshi Ohashi Dr. Irawati
Mr. Shoji Maehara Dr. Andria Agusta
Ms. Chinami Kitamura
Mr. Masakazu Tokuhisa
S y m b i o s i s r e l a t i o n s h i p between plant and
endophytic 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
Chemical compounds of mahkota dewa,
Phaleria macrocarpa

H3CO OR OH

OH O
R = H, benzophenone
HO Cycloargentenol
R = glucose, benzophenone HO -sitosterol

Bioactive compound CH2OH


HOH 2C O
O HO COOH
HO asam palmitat
CH2OH
HO O
OH COOH
OH
OH asam oleat
O OH
-D-glucopyranose COOH
asam linoleat
HOH 2C COOH
OH
OH asam linolenat

Sucrose
(-D-glucopyranosyl--D-fructofuranoside)
Produksi mikroba endofit C. aeruginosa (Jepang)

Produksi mikroba endofit C. domestica/C.longa (Indonesia)