The Japanese Traditional Applied Microbiology may Contribute to the - - PowerPoint PPT Presentation

The Japanese Traditional Applied Microbiology may Contribute to the Argentine Osao ADACHI & Kazunobu MATSUSHITA Laboratory of Applied Microbiology Department of Biological Chemistry Faculty of Agriculture, Yamaguchi University Yamaguchi 753-8515, Japan

Growth Habitats of Acetic Acid Bacteria

Flowers (Pistils/Pollens) Yeast, Fungi Lactic acid bacteria Zymomonas

In high conc. of sugars and sugar alcohols Competitive microbes Fruits and Flowers

(high sugars or sugar alcohols)

Fruit fly Vinegar fly Wine fly

Sushi based on Rice Vinegar

Static Rice Vinegar Fermentation

Cytoplasm Cytoplasm

PQQ

D-Glucose D-Gluconate

2-Keto-

D-gluconate

5-Keto-

D-gluconate

2,5-Diketo-

D-gluconate

PQQ PQQ FAD PQQ PQQ FAD PQQ PQQ PQQ MoCo? PQQ PQQ FAD? 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 Acetaldehyde Acetate Ribitol

L-Ribulose D-Fructose

5-Keto-D-fructose Quinate Dehydroquinate Ethanol

Cytoplasmic Cytoplasmic membrane membrane Outer surface Outer surface

Periplasm Periplasm

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・Gluconacetobacter

Physiological features Industrial development Basic Research Oxidative Fermentation

Vinegar Acetic acid Acetic acid fermentation fermentation Dehydroquinate fermentation Tartaric acid

(Chemicals)

Vitamin C

Acetic acid resistance

DHA

(Cosmetics)

Shikimate

(Medicine)

Fermentation-based Biotechnology of AAB Fermentation-based Biotechnology of AAB

5-Ketogluconate fermentation Dihydroxyacetone fermentation Ketogulonate fermentation Gluconobacter

D-Glucose D-Gluconate

2-Keto-

D-gluconate

5-Keto-

D-gluconate

2,5-Diketo-

D-gluconate L-Sorbose

Acetaldehyde

Quinate 3-Dehydroquinate

Ethanol Cytoplasmic Cytoplasmic membrane membrane Acetate

Periplasm Periplasm

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 OH H H HO C OH H C OH H C L-Sorbose CH 2OH CH2OH H HO C OH H C OH H C C O D-Glucose CHO C OH H CH 2OH H HO C OH H C OH H C COOH C OH H CH2OH H HO C OH H C OH H C D-Gluconate L-Sorbosone CHO CH 2OH H HO C O C OH H C C H HO

2-Keto-L-gulonate

HO COOH CH 2OH H HO C O C OH H 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/SNDH SNDH

Different Routes for Vitamin C

L-Ascorbate 5-Keto-D-gluconate

COOH C OH H CH 2OH H HO C OH H C O C L-Idonate COOH CH2OH H HO C OH H C C H HO OH H C

Gray's method Gray's method

COOH C O CH 2OH H HO C OH H C OH H C

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

COOH C O CH2OH H HO C OH H C O C

FAD-2KGADH In vitro in vitro enzymatic enzymatic FAD-SLDH

Reichstein-Grussner's method

D-Galactosamine D-Galactosaminate

CHO HCNH2 HOCH CH2OH HCOH HCOH COOH HCNH2 HOCH CH2OH HCOH HOCH

D-Mannosamine D-Mannosaminate

CH2OH CHO H2NCH HOCH HCOH HCOH COOH H2NCH HOCH CH2OH HCOH HCOH

D-Glucose D-Gluconate

CHO HCOH HOCH CH2OH HCOH HCOH COOH HCOH HOCH CH2OH HCOH HCOH

D-Glucosaminate D-Glucosamine

CH2OH CH2OH CHO HCNH2 HOCH HCOH HCOH COOH HCNH2 HOCH HCOH HCOH

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-P Phosphoenol- pyruvate 3-Deoxy-7-phospho-D- arabinoheptulosonate

Quinate

Dehydroquinate Dehydroshikimate

Shikimate

Ansamycin Rifamycin Chorismate Candicidin Nystatin Chloramphenicol Anthranilate Pyocyanine Tryptophan Actinomycin Prephenate Nocardicin Phenylalanine Polymyxin Tyrosine Novobiocin

QDH (Quinoprotein)

Metabolic Map of Shikimate Pathway Metabolic Map of Shikimate Pathway

COOH HO HO OH OH COOH HO O OH OH COOH O OH OH HO COOH OH OH

DQD SKDH

CH3 NH2 H N H H O O H3C O H CH3 O H3C

Oseltamivir (Tamiflu)

Nata de coco

Acetobacter lovaniensis NBRC 3284

Acetobacter aceti AJ 12368

Biocellulose and heteropolysaccharides of AAB Biocellulose 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 growth Biofirm Drug resistance

Absorbance at 660 nm Incubation period (min) Ferricyanide reduced (µmol) 0.2 0.4 20 40 60 80 0.1 0.2 0.05 µmol 0.1 µmol

Time Course of D-Fructose Oxidation

Time Course D-Gluconate Oxidation

Physiological features Industrial development Basic Research FDH-dependent battery

• K. Kano et al. (2007)

Oxidative fermentation PQQ・FAD Enzymes Biosensor Bio-fuel cell Cytoplasmic NAD(P) enzymes Chemicals・ Medicines

(Bioconversion)

AAB

Enzyme-based Biotechnology by using AAB Enzyme-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) Klett Ethanol, Acetic acid(g/100 ml)

• A. rancens SKU 1108

Acetate

Klett

Ethanol

• A. rancens IFO 3298
• A. aceti IFO 3283

6 4 2 4 2 4 2 250 150 50 150 50 200 100 40 80

Fermentation period (h)

(A) 30 C

Conversion rate (%) Klett units 24 48 24 48 72 40 80 120

(B) 37 C

100 200

growth 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 Imidazole acetaldehyde

Monoamine Ethylamine n-Propylamine n-Butylamine n-Amylamine n-Hexylamine Tyramine Tryptamine Epinephrine Norepinephrine Serotonin Agmatine Histamine 4.0 108.0 195.2 194.8 194.5 75.1 28.8 67.3 42.0 89.0 110.5 Monoamine Benzylamine Phenethylamine Diamine Ethylenediamine Propylenediamine Putrescine Cadaverine Hexylenediamine Polyamine Spermine Spermidine 100* 150.6 13.2 13.6 13.9 Substrate Relative rate 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）

COOH HO HO OH OH COOH HO O OH OH COOH O OH OH HO COOH OH OH

Dehydroquinate Dehydratase（DQD） Shikimate Dehydrogenase （SKDH）

CH3 NH2 H N H H O O H3C O H CH3 O H3C

Oseltamivir (Tamiflu)

3-Deoxyarabino- heptulosonate 7-phosphate

Coffee Pulp ＋

Caffeic acid Chlorogenate Hydrolase DOPA

Shikimic acid Pathway

Several Antibiotics Aromatic Amino acids Biodegradable Herbicide/Agrochemicals