3. gsc fukuyama

7/29/2019 3. GSC Fukuyama

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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-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


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)


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

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(-)-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)


(-)-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%)


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


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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


2

4

(+)-CA (1), (-)-EC (2),(-)-ECG (4), (-)-EGCG (5)


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-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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