Acetic acid bacteria, especially Gluconobacter species, have been known to catalyze the extensive oxidation of sugar alcohols (polyols) such as D-mannitol, glycerol, D-sorbitol, and so on. Gluconobacter species also oxidize sugars and sugar acids and uniquely accumulate two different keto-D-gluconates, 2-keto-D-gluconate and 5-keto-D-gluconate, in the culture medium by the oxidation of D-gluconate. However, there are still many controversies regarding their enzyme systems, especially on D-sorbitol and also D-gluconate oxidations. Recently, pyrroloquinoline quinone-dependent quinoprotein D-arabitol dehydrogenase and D-sorbitol dehydrogenase have been purified from G. suboxydans, both of which have similar and broad substrate specificity towards several different polyols. In this study, both quinoproteins were shown to be identical based on their immunocross-reactivity and also on gene disruption and were suggested to be the same as the previously isolated glycerol dehydrogenase (EC 1.1.99.22). Thus, glycerol dehydrogenase is the major polyol dehydrogenase involved in the oxidation of almost all sugar alcohols in Gluconobacter sp. In addition, the so-called quinoprotein glycerol dehydrogenase was also uniquely shown to oxidize D-gluconate, which was completely different from flavoprotein D-gluconate dehydrogenase (EC 1.1.99.3), which is involved in the production of 2-keto-Dgluconate. The gene disruption experiment and the reconstitution system of the purified enzyme in this study clearly showed that the production of 5-keto-D-gluconate in G. suboxydans is solely dependent on the quinoprotein glycerol dehydrogenase.Acetic acid bacteria are obligate aerobes well known as vinegar producers and also known to be able to oxidize various sugars and sugar alcohols such as D-glucose, glycerol, D-sorbitol, and so on, in addition to ethanol. Such oxidation reactions are called oxidative fermentation, since they involve incomplete oxidations of such alcohols or sugars accompanied by an accumulation of the corresponding oxidation products in large amounts in the culture medium. Of the two genera of acetic acid bacteria, Gluconobacter species extensively catalyze the oxidation of sugars and sugar alcohols except for ethanol, while Acetobacter species have a high ability to oxidize ethanol to acetic acid. These oxidation reactions of sugars or sugar alcohols seem to be carried out by membrane-bound dehydrogenases linked to the respiratory chain located in the cytoplasmic membrane of the organism (14). Of these oxidative fermentations of acetic acid bacteria, vinegar production from ethanol and 2-keto-D-gluconate (2KGA) production from glucose have each been shown to be carried out by sequential membranebound alcohol and aldehyde dehydrogenases and by glucose and gluconate dehydrogenases, respectively (14).There is still controversy about the mechanism of L-sorbose and 5-keto-D-gluconate (5KGA) production in Gluconobacter species. Three different membrane-bound enzymes have been proposed to be involved in L-sorbose production fr...

Diverse cellular processes such as autophagic protein degradation require phosphoinositide signaling in eukaryotic cells. In the methylotrophic yeast Pichia pastoris, peroxisomes can be selectively degraded via two types of pexophagic pathways, macropexophagy and micropexophagy. Both involve membrane fusion events at the vacuolar surface that are characterized by internalization of the boundary domain of the fusion complex, indicating that fusion occurs at the vertex. Here, we show that PpAtg24, a molecule with a phosphatidylinositol 3-phosphate-binding module (PX domain) that is indispensable for pexophagy, functions in membrane fusion at the vacuolar surface. CFP-tagged PpAtg24 localized to the vertex and boundary region of the pexophagosome-vacuole fusion complex during macropexophagy. Depletion of PpAtg24 resulted in the blockage of macropexophagy after pexophagosome formation and before the fusion stage. These and other results suggest that PpAtg24 is involved in the spatiotemporal regulation of membrane fusion at the vacuolar surface during pexophagy via binding to phosphatidylinositol 3-phosphate, rather than the previously suggested function in formation of the pexophagosome.

Glyceric acid (GA), an unfamiliar biotechnological product, is currently produced as a small by-product of dihydroxyacetone production from glycerol by Gluconobacter oxydans. We developed a method for the efficient biotechnological production of GA as a target compound for new surplus glycerol applications in the biodiesel and oleochemical industries. We investigated the ability of 162 acetic acid bacterial strains to produce GA from glycerol and found that the patterns of productivity and enantiomeric GA compositions obtained from several strains differed significantly. The growth parameters of two different strain types, Gluconobacter frateurii NBRC103465 and Acetobacter tropicalis NBRC16470, were optimized using a jar fermentor. G. frateurii accumulated 136.5 g/liter of GA with a 72% D-GA enantiomeric excess (ee) in the culture broth, whereas A. tropicalis produced 101.8 g/liter of D-GA with a 99% ee. The 136.5 g/liter of glycerate in the culture broth was concentrated to 236.5 g/liter by desalting electrodialysis during the 140-min operating time, and then, from 50 ml of the concentrated solution, 9.35 g of GA calcium salt was obtained by crystallization. Gene disruption analysis using G. oxydans IFO12528 revealed that the membrane-bound alcohol dehydrogenase (mADH)-encoding gene (adhA) is required for GA production, and purified mADH from G. oxydans IFO12528 catalyzed the oxidation of glycerol. These results strongly suggest that mADH is involved in GA production by acetic acid bacteria. We propose that GA is potentially mass producible from glycerol feedstock by a biotechnological process.

Phosphoinositides regulate a wide range of cellular activities, including membrane trafficking and biogenesis, via interaction with various effector proteins that contain phosphoinositide binding motifs. We show that in the yeast Pichia pastoris, phosphatidylinositol 4′-monophosphate (PI4P) initiates de novo membrane synthesis that is required for peroxisome degradation by selective autophagy and that this PI4P signaling is modulated by an ergosterol-converting PpAtg26 (autophagy-related) protein harboring a novel PI4P binding GRAM (glucosyltransferase, Rab-like GTPase activators, and myotubularins) domain. A phosphatidylinositol-4-OH kinase, PpPik1, is the primary source of PI4P. PI4P concentrated in a protein–lipid nucleation complex recruits PpAtg26 through an interaction with the GRAM domain. Sterol conversion by PpAtg26 at the nucleation complex is necessary for elongation and maturation of the membrane structure. This study reveals the role of the PI4P-signaling pathway in selective autophagy, a process comprising multistep molecular events that lead to the de novo membrane formation.

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