The Japanese Traditional Applied Microbiologymay Contribute to the Argentine

Osao ADACHI & Kazunobu MATSUSHITA

Laboratory of Applied MicrobiologyDepartment of Biological Chemistry

Faculty of Agriculture, Yamaguchi UniversityYamaguchi 753-8515, Japan

Growth Habitats of Acetic Acid Bacteria

Flowers (Pistils/Pollens)

Yeast, FungiLactic acid bacteria

Zymomonas

In high conc. of sugars and sugar alcohols Competitive microbes

Fruits and Flowers(high sugars or sugar alcohols)

Fruit flyVinegar flyWine fly

Sushi based on Rice Vinegar

Static Rice Vinegar Fermentation

CytoplasmCytoplasm

PQQ

D-Glucose D-Gluconate2-Keto-D-gluconate

5-Keto-D-gluconate

2,5-Diketo-D-gluconate

PQQ

PQQ

FAD

PQQ

PQQ

FAD

PQQ

PQQ

PQQ

MoCo? PQQ PQQFAD?

FAD PQQ FAD

D-Mannitol

D-Fructose

L-Sorbose

L-Sorbose

D-Sorbitol

D-Arabitol

D-Xylulose

Glycerol

meso-Erythritol

Dihydroxyacetone

L-Erythrulose

Cyclic alcohols

Cyclic ketones

Hexosamine

Hexosaminate

AcetaldehydeAcetate Ribitol

L-RibuloseD-Fructose

5-Keto-D-fructoseQuinate

DehydroquinateEthanol

CytoplasmicCytoplasmic membranemembrane

Outer surfaceOuter surface

PeriplasmPeriplasm

e- e- e- e-

e-

e-

e-

e- e- e- e- e- e-

e-

e-

e-

e-

D-Sorbitol

Cytosolic enzymes

Coenzymes (NAD/NADP, NADH/NADPH, PQQ, FAD, CoQ10)

Strong Enzymes in Acetic Acid Bacteria

Glucose dehydrogenase (NADP)2-Ketogluconate reductase (NADPH)5-Ketogluconate reductase (NADPH)Aldehyde dehydrogenase (NAD/NADP)Dioxyacetone reductase (NADH/NADPH)5-Ketofructose reductsae (NADPH)Shikimate dehydrogenase (NADP)Mannitol dehydrogenase (NADP)Sorbitol dehydrogenase (NAD)

Alcohol dehydrogenase (PQQ)Glucose dehydrogenase (PQQ)Gluconate dehydrogenase (FAD)Sorbitol dehydrogenase (PQQ)Glycerol dehydrogenase (PQQ)Aldehyde dehydrogenase (Mo)Fructose dehydrogenase (FAD)Quinate dehydrogenase (PQQ) Polyol dehydrogenase (PQQ)

Membrane-bound Cytoplasmic

Acetobacter・GluconacetobacterPhysiological features Industrial developmentBasic Research

Oxidative Fermentation

VinegarAcetic acid Acetic acid fermentationfermentation

Dehydroquinatefermentation

Tartaric acid(Chemicals)

Vitamin CAcetic acid resistance

DHA(Cosmetics)

Shikimate(Medicine)

Fermentation-based Biotechnology of AABFermentation-based Biotechnology of AAB

5-Ketogluconatefermentation

Dihydroxyacetonefermentation

Ketogulonatefermentation

Gluconobacter

D-Glucose D-Gluconate2-Keto-D-gluconate

5-Keto-D-gluconate

2,5-Diketo-D-gluconate

L-SorboseAcetaldehyde Quinate

3-DehydroquinateEthanol

CytoplasmicCytoplasmicmembranemembrane

Acetate

PeriplasmPeriplasm

e-e- e- e-

D-Sorbitol

Cytoplasm

UQ UQH2

e-e- e- e-

UQ UQH2

2H+

2H+ + O

H2O

2H+

2H+

Energy generation

Cyt. bo3

ADH ALDH SLDH QDH

GDH GADH GLDH 2KGDH

Membrane-bound Enzymes in Acetic Acid Bacteria and Energy Generation by Substrate Oxidation

D-Sorbitol

CH2OH

CH2OH

C OHH

HHO C

OHH C

OHH C

L-Sorbose

CH 2OH

CH2OH

HHO C

OHH C

OHH C

C O

D-Glucose

CHO

C OHH

CH 2OH

HHO C

OHH C

OHH C

COOH

C OHH

CH2OH

HHO C

OHH C

OHH C

D-Gluconate

L-Sorbosone

CHO

CH 2OH

HHO C

OC

OHH C

C HHO

2-Keto-L-gulonate

HO

COOH

CH 2OH

HHO C

OC

OHH C

C H

CO

HOC

HOC

HC

HOCH

O

CH 2OH

+H2/Cat

PQQ-GDH

PQQ-GLDH FAD-SDH

NAD(P)-SNDH

PQQ-GLDH

FAD-GADH

2,5-DKGR in vitro

PQQ-SDH/SNDH

PQQ-SDH/SNDHSNDH

Different Routes for Vitamin C

L-Ascorbate

5-Keto-D-gluconate

COOH

C OHH

CH 2OH

HHO C

OHH C

OC

L-Idonate

COOH

CH2OH

HHO C

OHH C

C HHO

OHH C Gray's methodGray's method

COOH

C O

CH 2OH

HHO C

OHH C

OHH C

2-Keto-D-gluconate 2,5-Diketo-D-gluconate

COOH

C O

CH2OH

HHO C

OHH C

OC

FAD-2KGADH

In vitro

in vitro

enzymatic

enzymatic

FAD-SLDH

Reichstein-Grussner's method

D-Galactosamine D-Galactosaminate

CHOHCNH2

HOCH

CH2OH

HCOHHCOH

COOHHCNH2

HOCH

CH2OHHCOH

HOCH

D-Mannosamine D-MannosaminateCH2OH

CHOH2NCHHOCH

HCOHHCOH

COOHH2NCHHOCH

CH2OHHCOHHCOH

D-Glucose D-Gluconate

CHOHCOH

HOCH

CH2OH

HCOHHCOH

COOHHCOH

HOCH

CH2OH

HCOHHCOH

D-GlucosaminateD-GlucosamineCH2OH CH2OH

CHOHCNH2

HOCHHCOHHCOH

COOHHCNH2

HOCHHCOHHCOH

Alternative Membrane-bound GDH from Gluconobacter

Hexosamine Oxidation

Rf

Glu N

Oxd Glu N

Man N

Gal N

Oxd Gal N

Oxd Man N

Glucosaminate

D-glucose

nojirimycin

1-deoxynojirimycin

Miglitol

OH O

OH O

OH

OH

O

O

Cyclobutanol Cyclobutanone

Cyclopentanol Cyclopentanone

1,3-Cyclopentanediol 1,3-Cyclopentanedione

OH

OH

OH O

O

O

OH O

Cyclohexanol Cyclohexanone

1,2-Cyclohexanediol 1,2-Cycloxanedione

Cyclooctanol Cyclooctanone

Cyclic Alcohol Oxidation

Glucose

Erythrose-4-PPhosphoenol-pyruvate

3-Deoxy-7-phospho-D-arabinoheptulosonate

Quinate

Dehydroquinate

Dehydroshikimate

ShikimateAnsamycinRifamycin

ChorismateCandicidinNystatin

Chloramphenicol

Anthranilate Pyocyanine

Tryptophan

ActinomycinPrephenateNocardicin

PhenylalaninePolymyxin Tyrosine Novobiocin

QDH (Quinoprotein)

Metabolic Map of Shikimate PathwayMetabolic Map of Shikimate Pathway

COOHHO

HOOH

OH

COOHHO

OOH

OH

COOH

OOH

OH

HO

COOH

OHOH

DQD

SKDH CH3

NH2

HNH H

OO

H3C

O

HCH3

OH3C

Oseltamivir (Tamiflu)

Nata de coco

Acetobacter lovaniensisNBRC 3284

Acetobacter aceti AJ 12368

Biocellulose and heteropolysaccharides of AABBiocellulose and heteropolysaccharides of AAB

（S. Yamanaka et al., J. Materials Science 24, 3141-3145, 1989） R strain

S strain

1 µm

Gluconacetobacter xylinus

Pellicle Static Aerobic growthBiofirm Drug resistance

Abs

orba

nce

at 6

60 n

m

Incubation period (min)

Ferr

icya

nide

redu

ced

(µm

ol)

0.2

0.4

20 40 60 800

0.1

0.2

0.05 µmol

0.1 µmol

Time Course of D-Fructose Oxidation

Time Course D-Gluconate Oxidation

Physiological features Industrial developmentBasic Research

FDH-dependent batteryK. Kano et al. (2007)

Oxidative fermentation

PQQ・FAD Enzymes

BiosensorBio-fuel cell

CytoplasmicNAD(P) enzymes

Chemicals・Medicines

(Bioconversion)

AAB

Enzyme-based Biotechnology by using AABEnzyme-based Biotechnology by using AAB

Gluconobacter

GDH

PQQ

FDH

FAD

Cyt c

n -hexanal

n -hexanal

Off-flavor Elimination from Soy Bean Meal with Acetic Acid Bacteria

Fermentation period (h)

Kle

tt

Etha

nol,

Ace

tic a

cid(

g/10

0 m

l)

A. rancens SKU 1108Acetate

Klett

Ethanol

A. rancens IFO 3298

A. aceti IFO 3283

6

4

2

0

4

2

0

4

2

0

250

150

50

0

150

50

0

200

100

00 40 80

Fermentation period (h)

(A) 30 C

Con

vers

ion

rate

(%)

Kle

tt un

its

0 24 48 0 24 48 720

40

80

120(B) 37 C

0

100

200growth

L-erythrulose

L-Erythrulose Production by Growing Cells of G. frateurii CHM 43

Fungal Strains for Koji Preparation and Rice Grains Covered with Asp. oryzae (Koji-gaku, H.Murakami(1985))

12 h

24 h

46 h

Asp. oryzae

Asp. oryzae

Asp. sojae

Rice Koji

Oxidation of Various Amines by Fungal Amine Oxidase

+ O2 + H2O2 + NH3

Histamine Imidazoleacetaldehyde

MonoamineEthylamine n-Propylaminen-Butylaminen-Amylaminen-HexylamineTyramineTryptamineEpinephrine NorepinephrineSerotoninAgmatineHistamine

4.0 108.0195.2194.8194.5 75.128.80

67.342.089.0

110.5

MonoamineBenzylaminePhenethylamine

DiamineEthylenediaminePropylenediaminePutrescineCadaverineHexylenediamine

PolyamineSpermineSpermidine

100*150.6

00

13.213.613.9

00

Substrate Relativerate Substrate Relative

rate

Fungal Tannase and Release of Polyphenols

Coffee Pulp Koji

Glucose

Erythrose 4-phosphate

Phosphoenolpyruvate

Quinate

3-Dehydroquinate

3-Dehydroshikimate

Shikimate

Quinate Dehydrogenase（QDH）

COOHHO

HOOH

OH

COOHHO

OOH

OH

COOH

OOH

OH

HO

COOH

OHOH

Dehydroquinate Dehydratase（DQD）

Shikimate Dehydrogenase （SKDH）

CH3

NH2

HNH H

OO

H3C

O

HCH3

OH3C

Oseltamivir (Tamiflu)

3-Deoxyarabino-heptulosonate7-phosphate

Coffee Pulp

＋ Caffeic acid

Chlorogenate Hydrolase

DOPA

Shikimic acid Pathway

Several Antibiotics AromaticAmino acids

Biodegradable Herbicide/Agrochemicals