#1The 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#2Growth Habitats of Acetic Acid Bacteria Fruits and Flowers (high sugars or sugar alcohols) Yeast, Fungi Lactic acid bacteria Zymomonas יוו Flowers (Pistils/Pollens) Competitive microbes In high conc. of sugars and sugar alcohols Fruit fly Vinegar fly Wine fly#3Sushi based on Rice Vinegar#4Static Rice Vinegar Fermentation#5D-Mannitol, D-Fructose D-Sorbitol L-Sorbose D-Sorbitol L-Sorbose D-Arabitol D-Xylulose D-Glucose D-Gluconate 2-Keto- D-gluconate PQQ PQQ FAD PQQ e PQQ FAD PQQ e e- e- 5-Keto- D-gluconate 2,5-Diketo- D-gluconate FAD e- Periplasm Outer surface Cytoplasmic membrane e Cytosolic enzymes e- e D e- e- PQQ MoCo? PQQ e- e- e e FAD? PQQ FAD PQQ PQQ PQQ Hexosamine Hexosaminate Glycerol Dihydroxyacetone meso-Erythritol L-Erythrulose Cyclic alcohols Cyclic ketones Ethanol Acetate Ribitol D-Fructose Quinate Acetaldehyde L-Ribulose 5-Keto-D-fructose Dehydroquinate#6Strong Enzymes in Acetic Acid Bacterial Membrane-bound 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) Cytoplasmic 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) Coenzymes (NAD/NADP, NADH/NADPH, PQQ, FAD, CoQ10)#7Fermentation-based Biotechnology of AAB Physiological features Basic Research Acetic acid resistance Industrial development Acetobacter Gluconacetobacter Acetic acid fermentation Vinegar Gluconobacter Oxidative Fermentation Ketogulonate fermentation Dihydroxyacetone fermentation Vitamin C DHA (Cosmetics) 5-Ketogluconate Tartaric acid fermentation (Chemicals) Dehydroquinate Shikimate fermentation (Medicine)#8Membrane-bound Enzymes in Acetic Acid Bacteria and Energy Generation by Substrate Oxidation 2-Keto- 5-Keto- 2,5-Diketo- D-Gluconate D-gluconate D-gluconate D-gluconate D-Glucose Periplasm GDH e Cytoplasmic UQ membrane UQ. e- ADH ALDH GADH GLDH e- (2KGDH e- e- UQH2 2H+ 2H+ + O Cytoplasm e- SLDH e- QDH 2H+ Cyt. bo H₂O 2H+ Energy UQH2 generation e- Quinate V 3-Dehydroquinate Ethanol Acetaldehyde D-Sorbitol L-Sorbose Acetate#9CH₂OH H-C-OH HO-C-H CH₂OH H-C-OH PQQ-GLDH HO-C-H CHO C=O FAD-SDH I HO-C-H H-C-OH FAD-SLDH H-C-OH H-C-OH I C=O H-C-OH PQQ-SDH/SNDH HO-C-H I SNDH CH₂OH CH₂OH CH₂OH +H2/Cat D-Sorbitol L-Sorbose Reichstein-Grussner's method In vitro L-Sorbosone CHO I H-C-OH HO-C-H 1 H-C-OH H-C-OH I CH₂OH PQQ-GDH D-Glucose COOH I H-C-OH I HO-C-H -----|--- H-C-OH I H-C-OH PQQ-SDH/SNDH NAD(P)-SNDH COOH I COOH COOH CO C = 0 C = O I FAD-2KGADH 2,5-DKGR HO-C-H I C=0 I HOC in vitro ။ FAD-GADH H-C-OH | H-C-OH CH2OH 2-Keto-D-gluconate HOCH H COH C=0 CH2OH 2,5-Diketo-D-gluconate HO-C-H | H-C-OH I HO-C-H I HOC enzymatic HC HOCH I CH2OH 2-Keto-L-gulonate CH2OH L-Ascorbate COOH COOH CH₂OH= H-C-OH 1 Gray's T H-C-OH Gray's method PQQ-GLDH D-Gluconate HOC H 1 HO-C-H Gray's method enzymatic in vitro H-C-OH I C=0 H-C-OH I HO-C-H 1 CH₂OH CH2OH 15-keto-D-gluconate L-Idonate Different Routes for Vitamin C#10CHO HCOH HOCH HCOH COOH HCOH CHO HCNH2 носн носн COOH HCNH2 носн нсон HCOH HOCH HCOH HCOH HCOH HCOH I CH2OH CH2OH CH2OH CH2OH D-Glucose D-Gluconate D-Galactosamine D-Galactosaminate CHO COOH CHO COOH HCNH2 HCNH2 H2NCH H2NCH HOCH носн носн HOCH HCOH нсон HCOH нсон HCOH HCOH HCOH HCOH CH2OH CH2OH CH2OH CH2OH D-Mannosaminate D-Glucosamine D-Glucosaminate D-Mannosamine Alternative Membrane-bound GDH from Gluconobacter#11Glu N Oxd Glu N Man N Oxd Man N Gal N Oxd Gal N Glucosaminate Rf Hexosamine Oxidation#12HO HO- HO HO- -OH D-glucose OH .OH H nojirimycin OH OH HO HO- OH H OH 1-deoxynojirimycin HO HO- OH -OH OH Miglitol OH#135-08-0 Cyclobutanol OH Cyclobutanone Cyclohexanol он OH Cyclohexanone 8-0 && Cyclopentanol Cyclopentanone 1,2-Cyclohexanediol 1,2-Cycloxanedione OH он 4-4 6-6 OH 1,3-Cyclopentanediol 1,3-Cyclopentanedione Cyclooctanol Cyclooctanone Cyclic Alcohol Oxidation#14Glucose Quinate Phosphoenol- Erythrose-4-P pyruvate Dehydroquinate 3-Deoxy-7-phospho-D- arabinoheptulosonate HO HO COOH OH OH QDH (Quinoprotein) HO COOH + DQD OF ON OH OH Dehydroshikimate SKDH COOH OH OH COOH Ansamycin Rifamycin -Shikimate HO OH OH Candicidin Nystatin Chorismate Anthranilate Chloramphenicol Nocardicin Prephenate Polymyxin Phenylalanine NH2 HIP CH 3 H3C N H H3C. CH3 Oseltamivir (Tamiflu) Pyocyanine Tryptophan Actinomycin Tyrosine Novobiocin Metabolic Map of Shikimate Pathway#15Nata de coco#16Biocellulose and heteropolysaccharides of AAB Pellicle Static Biofirm Aerobic growth Drug resistance Gluconacetobacter xylinus Acetobacter lovaniensis NBRC 3284 1 μπ Acetobacter aceti AJ 12368 (S. Yamanaka et al., J. Materials Science 24, 3141-3145, 1989) S strain 1111 38KU X29.980 1PM HD 8 1 um R strain#17Absorbance at 660 nm 0.4 0.1 μmol աղ 10.2 ㅎ 0.2 0.05 μmol 0.1 0 20 40 60 80 Incubation period (min) Time Course of D-Fructose Oxidation Ferricyanide reduced (μmol)#18Absorbance at 660 nm 0.6- 0.4 01030 0.2 0.0 300.0 Absorbance at 660 nm 0.6 0.4 0.2 0 0 0 0.05 0 0 20 0.10 Gluconate (umoles) 40 0.15 1 1 60 9310 Incubation period (min) Time Course D-Gluconate Oxidation#19Enzyme-based Biotechnology by using AAB Physiological features Basic Research AAB Gluconobacter PQQ GDH ●血値センサ・血 Cytoplasmic NAD(P) enzymes Oxidative fermentation PQQ •FAD Enzymes IIII HIEVEILLY Biosensor Bio-fuel cell FAD FDH Cyt c FDH-dependent battery K. Kano et al. (2007) Industrial development Chemicals • Medicines (Bioconversion)#20Recorder response A n-hexanal 2 4 6 80 2 B n-hexanal 4 6 8 Retention time (min) Off-flavor Elimination from Soy Bean Meal with Acetic Acid Bacteria#21Ethanol, Acetic acid (g/100 ml) 2 0 2 6 250 A. rancens SKU 1108 ○ Acetate 4 150 ■ Klett 2 ▲ Ethanol 50 A. rancens IFO 3298 150 50 0 A. aceti IFO 3283 200 40 80 Fermentation period (h) O 100 Klett#22Klett units ◇ 200- (A) 30°C growth 100- L-eryth rulose 1 0 24 48 0 24 (B) 37°C 120 48 72 Fermentation period (h) L-Erythrulose Production by Growing Cells of G. frateurii CHM 43 0 80 40 40 Conversion rate (%)#23Fungal Strains for Koji Preparation and Rice Grains Covered with Asp. oryzae (Koji-gaku, H.Murakami(1985)) Asp. oryzae 12 h Asp. oryzae 24 h Asp. sojae 46 h#24Rice Koji#25Oxidation of Various Amines by Fungal Amine Oxidase N + 02 2 + H,O, * NH3 NH2 Histamine Imidazole acetaldehyde Relative Relative Substrate Substrate rate rate Monoamine Monoamine Ethylamine 4.0 Benzylamine 100* n-Propylamine 108.0 Phenethylamine 150.6 n-Butylamine 195.2 Diamine n-Amylamine 194.8 Ethylenediamine 0 n-Hexylamine 194.5 Propylenediamine 0 Tyramine 75.1 Putrescine 13.2 Tryptamine 28.8 Cadaverine 13.6 Epinephrine 0 Hexylenediamine 13.9 Norepinephrine 67.3 Polyamine Serotonin 42.0 Spermine 0 Agmatine 89.0 Spermidine 0 Histamine 110.5#26HO Fungal Tannase and Release of Polyphenols HO HO HO HO HO HO OH HO HO HO 舌 HO. OH HO HO HO HO HO HO HO 。 CH 。 CH 天 . OH 全 天 西 CH 운 n=2~5 HO OH HO . OH OH OH -CH H OH OH H H OH HO OH -OH OH -OH#27Coffee Pulp Koji#28Phosphoenolpyruvate Coffee Pulp Chlorogenate Hydrolase Quinate HO COOH + Caffeic acid HO OH OH Quinate Dehydrogenase (QDH) DOPA HO COOH Glucose 3-Deoxyarabino- heptulosonate ✓ 7-phosphate 3-Dehydroquinate OH OH Dehydroquinate Dehydratase (DQD) Erythrose 4-phosphate Shikimic acid Pathway 3-Dehydroshikimate ☑ Shikimate COOH OH OH Shikimate Dehydrogenase (SKDH) COOH Several Antibiotics Aromatic Amino acids HO OH OH NH2 O HI CH 3 NI H3C H H H3C CH3 Biodegradable Oseltamivir (Tamiflu) Herbicide/Agrochemicals