Potassium ion leakage impairs thermotolerance in Corynebacterium glutamicum.

Corynebacterium glutamicum, a gram-positive bacterium, can produce amino acids such as glutamic acid and lysine. The heat generated during cell growth and/or glutamate fermentation disturbs both the cell growth and fermentation. To overcome such a negative effect of the fermentation heat, we have tried to establish a high temperature fermentation. One of the approach is to create a thermotolerant strains, while the other is to create an optimum culture conditions able for the strain to grow at higher temperatures. In this study, we focused on the latter approach, where we examined the effect of potassium ion on cell growth at high growth temperatures of C. glutamicum. The supplementation of high concentrations of potassium chloride (300 mM) (or sorbitol, an osmolyte) mitigated the repressed cell growth induced by high temperature at 39 °C or 40 °C. The intracellular potassium concentration declines from 300 mM to ∼150 mM by increasing the growth temperature but not by supplementing potassium chloride or sorbitol. Furthermore, in vitro experiments revealed that the potassium ion leakage occurs at high temperatures, which was mitigated in the presence of high concentrations of extracellular potassium chloride. This suggested that the presence of high osmolyte in the culture medium could inhibit the potassium ion leakage induced by high temperature and subsequently support cell growth at high temperatures.

Thermal adaptation of acetic acid bacteria for practical high-temperature vinegar fermentation.

Thermotolerant microorganisms are useful for high-temperature fermentation. Several thermally adapted strains were previously obtained from Acetobacter pasteurianus in a nutrient-rich culture medium, while these adapted strains could not grow well at high temperature in the nutrient-poor practical culture medium, "rice moromi." In this study, A. pasteurianus K-1034 originally capable of performing acetic acid fermentation in rice moromi was thermally adapted by experimental evolution using a "pseudo" rice moromi culture. The adapted strains thus obtained were confirmed to grow well in such the nutrient-poor media in flask or jar-fermentor culture up to 40 or 39 °C; the mutation sites of the strains were also determined. The high-temperature fermentation ability was also shown to be comparable with a low-nutrient adapted strain previously obtained. Using the practical fermentation system, "Acetofermenter," acetic acid production was compared in the moromi culture; the results showed that the adapted strains efficiently perform practical vinegar production under high-temperature conditions.

Change in product selectivity during the production of glyceric acid from glycerol by Gluconobacter strains in the presence of methanol.

To enhance the value-added use of methanol-containing raw glycerol derived from biodiesel fuel production, the effect of methanol supplementation on glyceric acid (GA) production by Gluconobacter spp. was investigated. We first conducted fed-batch fermentation with Gluconobacter frateurii NBRC103465 using raw glycerol as a feeding solution. GA productivity decreased with increasing dihydroxyacetone (DHA) formation when the raw glycerol contained methanol. The results of this experiment and comparative experiments using a synthetic solution modeled after the raw glycerol indicate that the presence of methanol caused a change in the concentrations of GA and DHA, two glycerol derivatives produced during fermentation. Other Gluconobacter spp. also decreased GA production in the presence of 1% (v/v) methanol. In addition, purified membrane-bound alcohol dehydrogenase (mADH) from Gluconobacter oxydans, which is a key enzyme in GA production, showed a decrease in dehydrogenase activity toward glycerol as the methanol concentration increased. These results strongly suggest that the observed decrease in GA production by Gluconobacter spp. resulted from the methanol-induced inhibition of mADH-mediated glycerol oxidation.

Crystallization and preliminary X-ray analysis of 5-keto-D-gluconate reductase from Gluconobacter suboxydans IFO12528 complexed with 5-keto-D-gluconate and NADPH.

NADPH-dependent 5-keto-D-gluconate reductase from Gluconobacter suboxydans IFO12528 (5KGR) catalyzes oxidoreduction between 5-keto-D-gluconate and D-gluconate with high specificity. 5KGR was expressed in Escherichia coli, purified and crystallized with 5-keto-D-gluconate and NADPH using the sitting-drop vapour-diffusion method at 288 K. A crystal of the 5KGR-NADPH complex was obtained using reservoir solution containing PEG 4000 as a precipitant and diffracted X-rays to 1.75 Šresolution. The crystal of the complex belonged to space group P4(2)2(1)2, with unit-cell parameters a=b=128.6, c=62.9 Å. A crystal of the 5KGR-NADPH-5-keto-D-gluconate complex was prepared by soaking the 5KGR-NADPH complex crystal in reservoir solution supplemented with 100 mM 5-keto-D-gluconate and 10 mM NADPH for 20 min and diffracted X-rays to 2.26 Šresolution. The crystal of the ternary complex belonged to space group P4(2)2(1)2, with unit-cell parameters a=b=128.7, c=62.5 Å. Both crystals contained two molecules in the asymmetric unit.

Siccanin rediscovered as a species-selective succinate dehydrogenase inhibitor.

To identify antibiotics targeting to respiratory enzymes, we carried out matrix screening of a structurally varied natural compound library with Pseudomonas aeruginosa membrane-bound respiratory enzymes. We identified a succinate dehydrogenase inhibitor, siccanin (IC(50), 0.9 microM), which is a potent antibiotic against some pathogenic fungi like Trichophyton mentagrophytes and inhibits their mitochondrial succinate dehydrogenase. We found that siccanin was effective against enzymes from P. aeruginosa, P. putida, rat and mouse mitochondria but ineffective or less effective against Escherichia coli, Corynebacterium glutamicum, and porcine mitochondria enzyme. Action mode was mixed-type for quinone-dependent activity and noncompetitive for succinate-dependent activity, indicating the proximity of the inhibitor-binding site to the quinone-binding site. Species-selective inhibition by siccanin is unique among succinate dehydrogenase inhibitors, and thus siccanin is a potential lead compound for new chemotherapeutics.

Identification of new inhibitors for alternative NADH dehydrogenase (NDH-II).

In bacterial membranes and plant, fungus and protist mitochondria, NADH dehydrogenase (NDH-II) serves as an alternative NADH : quinone reductase, a non-proton-pumping single-subunit enzyme bound to the membrane surface. Because NDH-II is absent in mammalian mitochondria, it is a promising target for new antibiotics. However, inhibitors for NDH-II are rare and unspecific. Taking advantage of the simple organization of the respiratory chain in Gluconobacter oxydans, we carried out screening of natural compounds and identified scopafungin and gramicidin S as inhibitors for G. oxydans NDH-II. Further, we examined their effects on Mycobacterium smegmatis and Plasmodium yoelii NDH-II as model pathogen enzymes.

Coffee pulp koji of Aspergillus sojae as stable immobilized catalyst of chlorogenate hydrolase.

Chlorogenate hydrolase (EC 3.1.1.42, CHase) was highly induced in mycelia of Aspergillus sojae AKU 3312 grown in Czapek medium containing either instant coffee powder or coffee pulp as inducer. No CHase formation was observed in the mycelia when cultivated without the inducer. CHase was purified readily from CHase-induced mycelia to high homogeneity, and the purified CHase revealed the molecular weight of 180,000 consisting of two identical subunits of 88 kDa. Equimolar quinate (QA) and caffeate (CA) were confirmed on hydrolysis of chlorogenate (CGA). The purified CHase was only useful for a laboratory scale hydrolysis of CGA. For practical QA and CA production using scaled up hydrolysis of vegetable extracts of natural CGA resources, the enzyme activity of purified CHase decreased and denatured irreversibly. Preparation of coffee pulp koji and its application to QA and CA production were proposed instead of purified CHase. When coffee pulp koji was heated at 60 degrees C for 30 min, CHase survived without any appreciable loss of enzyme activity while vegetative mycelial growth and spore germination were terminated. The heated coffee pulp koji thus prepared was effective itself as stable immobilized catalyst of CHase for QA and CA production from vegetable CGA resources such as coffee powders, coffee pulp, and others.