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 (eukaryota)

symbiosis or parasite

Microorganism

Endophyte (living inside of a plant) Epiphyte (living outside of a plant) Bacterium (prokaryota)

Fungus (eukaryota)

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 separation of colonies

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

separation of colonies

put on CMMA medium containing chloramphenicol (0.05 mg/ml)

inoculation onto PDA medium

Endophytic filamentous fungi from a tea plant were classified into six species by RAPD and rDNA sequences analysis
fungus A B C D E F family name Nectriaceae Trichocomaceae Schizophyllaceae Schizophyllaceae Valsaceae Valsaceae genus name Fusarium Penicillium Schizophyllum Schizophyllum Diaporthe Diaporthe accession no.* AB245442 AB245443 AB245444 AB245445 AB245446 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 HO O OH OH OH HO O OH OH OH R

(+)-catechin (1, CA)

(-)-epicatechin R = H (2, EC) (-)-epigallocatechin R = OH (3, EGC)

OH OH OH HO O OH O galloyl OH

OH HO O O galloyl 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 2. Nutrient broth with 0.5% NaCl 3. 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

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 (20 mg) in MeOH

Diaporthe sp. (fungus E)

200 ml of glucose-yeast extract-peptone

biotransformed product (9.5 mg, 45%)

1) cultivation for one day 2) extraction with EtOAc 1) SiO2 column

(CHCl3:MeOH:H2O=65:35:10, lower phase)

2) HPLC (Capcell Pak Phenyl recovered (+)-catechin (1.7 mg, 8.5%)

SG-120, 250 x 4.6 mm, H2O)

EtOAc-soluble portion (45 mg)

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 time (min) 30.00 0.00

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 OH HO O R
4 2 3 S

C15H14O7 (HR-FABMS) []D +5.5o (EtOH) IR (KBr): 3300, 1612 cm-1 UV (EtOH): 280 nm (= 4.000)

OH

OH

OH

(+)-(2R,3S,4S)-3,4,5,7,3',4'hexahydroxyflavan (6)*
OH OH HO O OH OH

H NMR (d6-acetone) 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)

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

(+)-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

OH OH HO O R R OH OH Diaporthe sp. (fungus E) HO

nOe
H O H 2 H 4 R R HO OH

39%

HO

(-)-epicatechin (2)

(-)-(2R,3R,4R)-3,4,5,7,3',4'hexahydroxyflavan (7, new)

. 7: C15H14O7 (HR-FABMS) o []D -8.4 (EtOH) IR (KBr): 3250, 1628 cm-1 UV (EtOH): 279 nm ( = 4,200)

H 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)

OH OH HO O R R OH OH Diaporthe sp. (fungus E) O R

OH OH HO

39%
OH OH

R 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 absorbance

biotransformed product (7, 39%)

HO O OH OH

OH

(-)-epicatechin (2, 2.4%)

0.00 0.00 10.00 20.00 time (min) 30.00 0.00

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 HO OR
R

OH OH HO OR
R

OH OH HO OR
R 2 R 43

Diaporthe sp O-galloyl (fungus E)

OH OH
(-)-epicatechin (2)

OH

OH
(-)-epicatechin 3-O-gallate (4)

OH OH
(-)-(2R,3R,4R)-3,4,5,7,3',4'hexahydroxyflavan (7, 53%)

OH OH HO OR
R

OH OH HO OR
R

OH OH HO
R

OH Diaporthe sp O-galloyl (fungus E)

OH OH

OR

2 4 3R

OH OH

OH
(-)-epigallocatechin 3-O-gallate (5)

OH
(-)-epigallocatechin (3)

OH OH
(-)-(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

200.00

OH

biotransformed product (7, 53%)

absorbance

HO

O O galloyl OH

(-)-ECG (4) (-)-epicatechin (2, 13%)

0.00 0.00 10.00

20.00
time (min)

30.00

40.00

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

biotransformed product (8, 43%)

HO

OH O galloyl

absorbance

OH

(-)-epigallocatechin (3, 19%)

(-)-EGCG (5)

0.00 0.00 10.00 20.00 time (min) 30.00 40.00

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 HO O
4 3 S

OH OH
Diaporthe sp. (fungus E)

HO

O
4 3S

OH

OH

OH 3S : (+)-CA (1)

OH OH Insertion of 4S-hydroxyl group [(+)-(2R,3S,4S)-3,4,5,7,3',4'hexahydroxyflavan (6)] OH OH OH OH

Diaporthe sp. (fungus E)

HO

O
4 3 R

R2

HO

O
4 3 R

R2

OR1

OH

OH 3R : (-)-EC (2); (-)-ECG (4) (-)-EGCG (5)

OH OH Insertion of 4R-hydroxyl group [(-)-(2R,3R,4R)-3,4,5,7,3',4'hexahydroxyflavans (7 and 8)]

Substrate specificity
2R
HO OR
2

OH OH R2 OR1 OH (+)-CA (1), (-)-EC (2), (-)-ECG (4), (-)-EGCG (5) oxidation at C-4 Diaporthe sp. (fungus E) HO OR
2 4

OH OH R2 OH

OH OH 3,4-cis-dihydroxyflavan (6,7,8)

2S
HO OS
2

OH OH OH O galloyl hydrolysis (-)-GC (17) HO OS

2

OH OH recovered

OH OH

OH (-)-GCG (9)

Diaporthe sp. (fungus E)

(-)-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

OH OH HO O OH OH

Dendrophthoe pentandra (Stem and leaves)

1) extracted with 70% aq.acetone 2) evap. in vacuo

70% aq.acetone extract

partitioned into EtOAc and water

(+)-catechin

EtOAc-soluble portion
SiO2 column chr.

H2O soluble portion

Fr. 1

Fr. 2

Fr. 3 (+)-catechin

Fr. 4
HPLC

Identified by []D, UV, IR and NMR

Screening test of an ability to bio-produce (+)-catechin

parasitic plant Dendrophthoe pentandra

Separation

tea plant Camellia sinensis

Mediums used for screening tests

2 fungi (G and H)

1. 2. 3. 4.

Phoma medium S7 medium Glucose-peptone medium PDB

6 fungi (A ~ F)

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
Fungus H, a new fungus, Phomopsis sp.
0.06 0.05 0.04 0.03 0.02 0.01 0.00 0 5 10 15 20 25 30 35 40 45 (min)

Bio-products

Identified by []D, UV, IR, NMR (+)-catechin

CAPCELL PAK C18, 20 mM Phosphate buffer-acetonitrile (gradient), 210 nm

What`s the genuine producer of (+)-catechin ?

Tea plant
Camellia sinensis (L.) O.K.

Parasitic plant
Dendrophthoe pentandra

Production ?? separation
OH OH HO O OH OH

production

Endophyte 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 biosynthesize (+)-catechin

Law of Applied Microbiology Law of Applied Microbiology

- The microorganism isis always right, - The microorganism always right, your friend and sensitive partner your friend and sensitive partner - They are no stupid microorganism - They are no stupid microorganism

- Microorganisms can (will) do anything - Microorganisms can (will) do anything

- Microorganism are smarter, wiser, more - Microorganism are smarter, wiser, more energetic than chemist, microbiologist, etc energetic than chemist, microbiologist, etc - If If you take of your (microbial) friends, they - you take of your (microbial) friends, they will take care ofof your future will take care your future (and you will live happily everyafter) (and you will live happily everyafter)
(D. Parlman, 1979)

The Puncak area, West Java, Indonesia

Collaboration between Fukuyama University and Indonesian Institute of Sciences

Fukuyama University
Dr. Hirotaka Shibuya Dr. Kazuyoshi Ohashi Mr. Shoji Maehara Ms. Chinami Kitamura Mr. Masakazu Tokuhisa

Indonesian Institute of Sciences

Dr. Made Sri Prana Dr. Irawati Dr. Andria Agusta

 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 HO Cycloargentenol CH2OH O O OH OH HO O OH O OH CH2OH COOH asam palmitat COOH asam oleat COOH asam linoleat HOH2C OH OH COOH asam linolenat HO -sitosterol

R = H, benzophenone R = glucose, benzophenone

Bioactive compound
HOH2C HO HO

-D-glucopyranose

Sucrose (-D-glucopyranosyl--D-fructofuranoside)

Produksi mikroba endofit C. aeruginosa (Jepang)

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