Implication of amino acid metabolism and cell surface integrity for the thermotolerance mechanism in the thermally adapted acetic acid bacterium Acetobacter pasteurianus TH-3.

IMPORTANCE: Acetobacter pasteurianus, an industrial vinegar-producing strain, is suffered by fermentation stress such as fermentation heat and/or high concentrations of acetic acid. By an experimental evolution approach, we have obtained a stress-tolerant strain, exhibiting significantly increased growth and acetic acid fermentation ability at higher temperatures. In this study, we report that only the three gene mutations of ones accumulated during the adaptation process, ansP, dctD, and glnD, were sufficient to reproduce the increased thermotolerance of A. pasteurianus. These mutations resulted in cell envelope modification, including increased phospholipid and lipopolysaccharide synthesis, increased respiratory activity, and cell size reduction. The phenotypic changes may cooperatively work to make the adapted cell thermotolerant by enhancing cell surface integrity, nutrient or oxygen availability, and energy generation.

Development of a 2-hydroxyglutarate production system by Corynebacterium glutamicum.

2-Oxoglutarate (2-OG) is a tricarboxylate cycle intermediate that can be biologically converted into several industrially important compounds. However, studies on the fermentative production of compounds synthesized from 2-OG, but not via glutamate (defined as 2-OG derivatives), have been limited. Herein, a system that can efficiently produce 2-hydroxyglutarate (2-HG), a 2-OG derivative biosynthesized by the hgdH-encoded NADH-dependent 2-HG dehydrogenase of Acidaminococcus fermentans, was developed as a model using Corynebacterium glutamicum. First, the D3 strain, which lacked the two NADH-consuming enzymes, lactate dehydrogenase and malate dehydrogenase, as well as isocitrate lyase, was constructed as a starting strain. Next, the growth conditions that induced the accumulation of 2-OG were investigated, and it was found that the biotin- and nitrogen-limited (B/N-limited) aerobic growth conditions were suitable for this purpose. Finally, the hgdH gene of A. fermentans became overexpressed in the D3 strain by inserting it into the intergenic regions with the strong constitutive promoter of the tuf gene of C. glutamicum; the engineered strain was cultured under the B/N-limited aerobic growth conditions. The engineered strain produced 80.1 mM 2-HG with a yield of 0.390 mol/mol glucose, which are the highest titer and yield reported thus far, to the best of our knowledge. Furthermore, reverse genetics showed that the produced 2-HG was partially exported via the YggB protein (NCgl1221 protein, a mechanosensitive channel) known as an exporter for glutamate under the conditions used herein. KEY POINTS: • An efficient 2-HG production system was developed with Corynebacterium glutamicum. • Biotin- and nitrogen-limited aerobic growth conditions induced 2-OG production. • Produced 2-HG was partially excreted via the glutamate exporter, YggB.

Dissection and Reconstitution Provide Insights into Electron Transport in the Membrane-Bound Aldehyde Dehydrogenase Complex of Gluconacetobacter diazotrophicus.

Acetic acid bacteria catalyze the two-step oxidation of ethanol to acetic acid using the membrane-bound enzymes pyrroloquinoline quinone-dependent alcohol dehydrogenase and molybdopterin-dependent aldehyde dehydrogenase (ALDH). Although the reducing equivalents from the substrate are transferred to ubiquinone in the membrane, intramolecular electron transport in ALDH is not understood. Here, we purified the AldFGH complex, the membrane-bound ALDH that is physiologically relevant to acetic acid fermentation in Gluconacetobacter diazotrophicus strain PAL5. The purified AldFGH complex showed acetaldehyde:ubiquinone (Q2) oxidoreductase activity. c-type cytochromes of the AldFGH complex (in the AldF subunit) were reduced by acetaldehyde. Next, we genetically dissected the AldFGH complex into AldGH and AldF units and reconstituted them. The AldGH subcomplex showed acetaldehyde:ferricyanide oxidoreductase activity but not Q2 reductase activity. The ALDH activity of AldGH was not found in membranes but was found in the soluble fraction of the recombinant strain, suggesting that the AldF subunit is responsible for membrane binding of the AldFGH complex. The absorption spectra of the purified AldGH subcomplex suggested the presence of an [Fe-S] cluster, which can be reduced by acetaldehyde. The AldFGH complex reconstituted from the AldGH subcomplex and AldF showed Q2 reductase activity. We propose a model in which electrons from the substrate are abstracted by a molybdopterin in the AldH subunit and transferred to the [Fe-S] cluster(s) in the AldG subunit, followed by electron transport to c-type cytochrome centers in the AldF subunit, which is the site of ubiquinone reduction in the membrane. IMPORTANCE Two membrane-bound enzymes of acetic acid bacteria, pyrroloquinoline quinone-dependent alcohol dehydrogenase and molybdopterin-dependent aldehyde dehydrogenase (ALDH), are responsible for vinegar production. Upon the oxidation of acetaldehyde, ALDH reduces ubiquinone in the cytoplasmic membrane. ALDH is an enzyme complex of three subunits. Here, we tried to understand how ALDH works by using a classical biochemical approach and genetic engineering to dissect the enzyme complex into soluble and membrane-bound parts. The soluble part had limited activity in vitro and did not reduce ubiquinone. However, the enzyme complex reconstituted from the soluble and membrane-bound parts showed ubiquinone reduction activity. The proposed working model of ALDH provides a better understanding of how the enzyme works in the vinegar fermentation process.

Development of efficient 5-ketogluconate production system by Gluconobacter japonicus.

5-Ketogluconate (5KGA) is a precursor for synthesizing tartrate, a valuable compound used in several industries. In a previous study, Gluconobacter japonicus NBRC 3271 mutant strain D2, which lacks two membranous gluconate 2-dehydrogenases, was shown to produce 5KGA but not 2-ketogluconate from a mixture of glucose and gluconate. In this study, we aimed to develop an efficient 5KGA production system using G. japonicus D2 as the parental strain. D2 produced 5KGA from glucose in a jar fermentor culture; however, 5KGA levels were reduced during the late phase of cultivation. To increase the potential of D2 for 5KGA production, the cytoplasmic metabolism related to the utilization of 5KGA and gluconate was modified; the gno and gntK genes encoding 5KGA reductase and gluconokinase, respectively, were deleted from D2, generating D4. Improved 5KGA production was observed in D4 compared to that in D2, but a significant amount of gluconate remained at the end of cultivation, leading to an unsatisfied yield of 0.83 mol (mol glucose)-1. The conversion of gluconate to 5KGA is catalyzed by pyrroloquinoline quinone (PQQ)-dependent glycerol dehydrogenase (GLDH), which easily forms an apoenzyme by releasing PQQ and calcium ions. Thus, the effects of CaCl2 addition to the culture medium on 5KGA production by D4 were investigated. We demonstrated that 1 mM CaCl2 addition positively affected the maintenance of the PQQ-GLDH activity toward gluconate and consequently enhanced 5KGA production, and the yield reached 0.97 mol (mol glucose)-1. KEY POINTS: • An efficient 5KGA production system was developed with Gluconobacter japonicus. • Deleting the gno and gntK genes blocked the catabolism of 5KGA and gluconate. • The addition of 1 mM CaCl2 efficiently improved the conversion of glucose to 5KGA.

Membrane-bound D-mannose isomerase of acetic acid bacteria: finding, characterization, and application.

D-Mannose isomerase (EC 5.3.1.7) catalyzing reversible conversion between D-mannose and D-fructose was found in acetic acid bacteria. Cell fractionation confirmed the enzyme to be a typical membrane-bound enzyme, while all sugar isomerases so far reported are cytoplasmic. The optimal enzyme activity was found at pH 5.5, which was clear contrast to the cytoplasmic enzymes having alkaline optimal pH. The enzyme was heat stable and the optimal reaction temperature was observed at around 40 to 60˚C. Purified enzyme after solubilization from membrane fraction showed the total molecular mass of 196 kDa composing of identical four subunits of 48 kDa. Washed cells or immobilized cells were well functional at nearly 80% of conversion ratio from D-mannose to D-fructose and reversely 20-25% of D-fructose to D-mannose. Catalytic properties of the enzyme were discussed with respect to the biotechnological applications to high fructose syrup production from konjac taro.

Characterization of 3 phylogenetically distinct membrane-bound d-gluconate dehydrogenases of Gluconobacter spp. and their biotechnological application for efficient 2-keto-d-gluconate production.

We identified a novel flavoprotein-cytochrome c complex d-gluconate dehydrogenase (GADH) encoded by gndXYZ of Gluconobacter oxydans NBRC 3293, which is phylogenetically distinct from previously reported GADHs encoded by gndFGH and gndSLC of Gluconobacter spp. To analyze the biochemical properties of respective GADHs, Gluconobacter japonicus NBRC 3271 mutant strain lacking membranous d-gluconate dehydrogenase activity was constructed. All GADHs (GndFGH, GndSLC, and GndXYZ) were successfully overexpressed in the constructed strain. The optimal pH and KM value at that pH of GndFGH, GndSLC, and GndXYZ were 5, 6, and 4, and 8.82 ± 1.15, 22.9 ± 5.0, and 11.3 ± 1.5 m m, respectively. When the mutants overexpressing respective GADHs were cultured in d-glucose-containing medium, all of them produced 2-keto-d-gluconate, revealing that GndXYZ converts d-gluconate to 2-keto-d-gluconate as well as other GADHs. Among the recombinants, the gndXYZ-overexpressing strain accumulated the highest level of 2-keto-d-gluconate, suggesting its potential for 2-keto-d-gluconate production.

Periplasmic dehydroshikimate dehydratase combined with quinate oxidation in Gluconobacter oxydans for protocatechuate production.

Protocatechuate (3,4-dihydroxybenzoate) has antioxidant properties and is a raw material for the production of muconic acid, which is a key compound in the synthesis of polymers such as nylon and polyethylene terephthalate. Gluconobacter oxydans strain NBRC3244 has a periplasmic system for oxidation of quinate to produce 3-dehydroquinate. Previously, a periplasmic 3-dehydroshikimate production system was constructed by heterologously expressing Gluconacetobacter diazotrophicus dehydroquinate dehydratase in the periplasm of G. oxydans strain NBRC3244. 3-Dehydroshikimate is converted to protocatechuate by dehydration. In this study, we constructed a G. oxydans strain that expresses the Acinetobacter baylyi quiC gene, which encodes a dehydroshikimate dehydratase of which the subcellular localization is likely the periplasm. We attempted to produce protocatechuate by co-cultivation of two recombinant G. oxydans strains-one expressing the periplasmically targeted dehydroquinate dehydratase and the other expressing A. baylyi dehydroshikimate dehydratase. The co-cultivation system produced protocatechuate from quinate in a nearly quantitative manner.

Histamine elimination by a coupling reaction of fungal amine oxidase and bacterial aldehyde oxidase†.

Histamine (HIST) and other biogenic amines found in fish and fishery products accumulated by the action of bacterial amino acid decarboxylase cannot be decomposed and eliminated by heating or other chemical methods. A simple method for HIST elimination is proposed by a coupling reaction of the fungal amine oxidase (FAO) and bacterial aldehyde oxidase (ALOX) of acetic acid bacteria. As a model reaction, FAO oxidized benzylamine to benzaldehyde, which in turn was oxidized spontaneously to benzoic acid with ALOX. Likely, in HIST elimination, FAO coupled well with ALOX to produce imidazole 4-acetic acid from HIST with an apparent yield of 100%. Imidazole 4-acetaldehyde was not detected in the reaction mixture. In the absence of ALOX, the coupling reaction was incomplete given a number of unidentified substances in the reaction mixture. The proposed coupling enzymatic method may be highly effective to eliminate toxic amines from fish and fishery products.

The 5-Ketofructose Reductase of Gluconobacter sp. Strain CHM43 Is a Novel Class in the Shikimate Dehydrogenase Family.

Gluconobacter sp. strain CHM43 oxidizes mannitol to fructose and then oxidizes fructose to 5-keto-d-fructose (5KF) in the periplasmic space. Since NADPH-dependent 5KF reductase was found in the soluble fraction of Gluconobacter spp., 5KF might be transported into the cytoplasm and metabolized. Here, we identified the GLF_2050 gene as the kfr gene encoding 5KF reductase (KFR). A mutant strain devoid of the kfr gene showed lower KFR activity and no 5KF consumption. The crystal structure revealed that KFR is similar to NADP+-dependent shikimate dehydrogenase (SDH), which catalyzes the reversible NADP+-dependent oxidation of shikimate to 3-dehydroshikimate. We found that several amino acid residues in the putative substrate-binding site of KFR were different from those of SDH. Phylogenetic analyses revealed that only a subclass in the SDH family containing KFR conserved such a unique substrate-binding site. We constructed KFR derivatives with amino acid substitutions, including replacement of Asn21 in the substrate-binding site with Ser that is found in SDH. The KFR-N21S derivative showed a strong increase in the Km value for 5KF but a higher shikimate oxidation activity than wild-type KFR, suggesting that Asn21 is important for 5KF binding. In addition, the conserved catalytic dyad Lys72 and Asp108 were individually substituted for Asn. The K72N and D108N derivatives showed only negligible activities without a dramatic change in the Km value for 5KF, suggesting a catalytic mechanism similar to that of SDH. With these data taken together, we suggest that KFR is a new member of the SDH family. IMPORTANCE A limited number of species of acetic acid bacteria, such as Gluconobacter sp. strain CHM43, produce 5-ketofructose, a potential low-calorie sweetener, at a high yield. Here, we show that an NADPH-dependent 5-ketofructose reductase (KFR) is involved in 5-ketofructose degradation, and we characterize this enzyme with respect to its structure, phylogeny, and function. The crystal structure of KFR was similar to that of shikimate dehydrogenase, which is functionally crucial in the shikimate pathway in bacteria and plants. Phylogenetic analysis suggested that KFR is positioned in a small subgroup of the shikimate dehydrogenase family. Catalytically important amino acid residues were also conserved, and their relevance was experimentally validated. Thus, we propose KFR as a new member of shikimate dehydrogenase family.

The Auxiliary NADH Dehydrogenase Plays a Crucial Role in Redox Homeostasis of Nicotinamide Cofactors in the Absence of the Periplasmic Oxidation System in Gluconobacter oxydans NBRC3293.

Gluconobacter oxydans has the unique property of a glucose oxidation system in the periplasmic space, where glucose is oxidized incompletely to ketogluconic acids in a nicotinamide cofactor-independent manner. Elimination of the gdhM gene for membrane-bound glucose dehydrogenase, the first enzyme for the periplasmic glucose oxidation system, induces a metabolic change whereby glucose is oxidized in the cytoplasm to acetic acid. G. oxydans strain NBRC3293 possesses two molecular species of type II NADH dehydrogenase (NDH), the primary and auxiliary NDHs that oxidize NAD(P)H by reducing ubiquinone in the cell membrane. The substrate specificities of the two NDHs are different from each other: primary NDH (p-NDH) oxidizes NADH specifically but auxiliary NDH (a-NDH) oxidizes both NADH and NADPH. We constructed G. oxydans NBRC3293 derivatives defective in the ndhA gene for a-NDH, in the gdhM gene, and in both. Our ΔgdhM derivative yielded higher cell biomass on glucose, as reported previously, but grew at a lower rate than the wild-type strain. The ΔndhA derivative showed growth behavior on glucose similar to that of the wild type. The ΔgdhM ΔndhA double mutant showed greatly delayed growth on glucose, but its cell biomass was similar to that of the ΔgdhM strain. The double mutant accumulated intracellular levels of NAD(P)H and thus shifted the redox balance to reduction. Accumulated NAD(P)H levels might repress growth on glucose by limiting oxidative metabolisms in the cytoplasm. We suggest that a-NDH plays a crucial role in redox homeostasis of nicotinamide cofactors in the absence of the periplasmic oxidation system in G. oxydansIMPORTANCE Nicotinamide cofactors NAD+ and NADP+ mediate redox reactions in metabolism. Gluconobacter oxydans, a member of the acetic acid bacteria, oxidizes glucose incompletely in the periplasmic space-outside the cell. This incomplete oxidation of glucose is independent of nicotinamide cofactors. However, if the periplasmic oxidation of glucose is abolished, the cells oxidize glucose in the cytoplasm by reducing nicotinamide cofactors. Reduced forms of nicotinamide cofactors are reoxidized by NADH dehydrogenase (NDH) on the cell membrane. We found that two kinds of NDH in G. oxydans have different substrate specificities: the primary enzyme is NADH specific, and the auxiliary one oxidizes both NADH and NADPH. Inactivation of the latter enzyme in G. oxydans cells in which we had induced cytoplasmic glucose oxidation resulted in elevated intracellular levels of NAD(P)H, limiting cell growth on glucose. We suggest that the auxiliary enzyme is important if G. oxydans grows independently of the periplasmic oxidation system.

Heterologous expression of membrane-bound alcohol dehydrogenase-encoding genes for glyceric acid production using Gluconobacter sp. CHM43 and its derivatives.

In contrast to D-glyceric acid (D-GA) production with 99% enantiomeric excess (ee) by Acetobacter tropicalis NBRC 16470, Gluconobacter sp. CHM43 produced 19.6 g L-1 of D-GA with 73.7% ee over 4 days of incubation in flask culture. To investigate the reason for this enantiomeric composition of GA, the genes encoding membrane-bound alcohol dehydrogenase (mADH) of A. tropicalis NBRC 16470, composed of three subunits (adhA, adhB, and adhS), were cloned using the broad-host-range vector pBBR1MCS-2 and heterologously expressed in Gluconobacter sp. CHM43 and its ΔadhAB ΔsldBA derivative TORI4. Reverse-transcription quantitative real-time polymerase chain reaction demonstrated that adhABS genes from A. tropicalis were expressed in TORI4 transformants, and their membrane fraction exhibited mADH activities of 0.13 and 0.31 U/mg with or without AdhS, respectively. Compared with the GA production of TORI4-harboring pBBR1MCS-2 (1.23 g L-1), TORI4 transformants expressing adhABS and adhAB showed elevated GA production of 2.46 and 3.67 g L-1, respectively, suggesting a negative effect of adhS gene expression on GA production as well as mADH activity in TORI4. Although TORI4 was found to produce primarily L-GA with 42.5% ee, TORI4 transformants expressing adhABS and adhAB produced D-GA with 27.6% and 49.0% ee, respectively, demonstrating that mADH of A. tropicalis causes a sharp increase in the enantiomeric composition of D-GA. These results suggest that one reason for D-GA production with 73.7% ee in Gluconobacter spp. might be a property of the host, which possibly produces L-GA intracellularly. KEY POINTS: • Membrane-bound ADH from Acetobacter tropicalis showed activity in Gluconobacter sp. • D-GA production from glycerol was performed using recombinant Gluconobacter sp. • Enantiomeric excess of D-GA was affected by both membrane and intracellular ADHs.

Major aldehyde dehydrogenase AldFGH of Gluconacetobacter diazotrophicus is independent of pyrroloquinoline quinone but dependent on molybdopterin for acetic acid fermentation.

Acetic acid fermentation involves the oxidation of ethanol to acetic acid via acetaldehyde as the intermediate and is catalyzed by the membrane-bound alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) of acetic acid bacteria. Although ADH depends on pyrroloquinoline quinone (PQQ), the prosthetic group associated with ALDH remains a matter of debate. This study aimed to address the dependency of ALDH of Gluconacetobacter diazotrophicus strain PAL5 on PQQ and the physiological role of ALDH in acetic acid fermentation. We constructed deletion mutant strains for both the ALDH gene clusters of PAL5, aldFGH and aldSLC. In addition, the adhAB operon for ADH was eliminated, since it shows ALDH activity. The triple-deletion derivative ΔaldFGH ΔaldSLC ΔadhAB failed to show ALDH activity, which suggested that ALDH activity in PAL5 is derived from these three enzyme complexes. Since the single-gene cluster deletion derivative ΔaldFGH lost most ALDH activity, and accumulated much higher acetaldehyde than wild type under acetic acid fermentation conditions, we concluded that AldFGH functions as the major ALDH in PAL5. Furthermore, deletion of the PQQ biosynthesis gene cluster (pqqABCDE) abolished ADH activity completely, but did not affect ALDH activity. Instead, the molybdopterin biosynthesis gene deletion derivatives lost ALDH activity. Thus, we concluded that the AldFGH and AldSLC complexes of Ga. diazotrophicus PAL5 require a form of molybdopterin but not PQQ for ALDH activity. KEY POINTS: • AldFGH is the major aldehyde dehydrogenase in Gluconacetobacter diazotrophicus PAL5. • Acetaldehyde accumulated from ethanol in the absence of AldFGH. • Molybdopterin, rather than pyrroloquinoline quinone, is required for AldFGH.

Three ATP-dependent phosphorylating enzymes in the first committed step of dihydroxyacetone metabolism in Gluconobacter thailandicus NBRC3255.

Dihydroxyacetone (DHA), a chemical suntan agent, is produced by the regiospecific oxidation of glycerol with Gluconobacter thailandicus NBRC3255. However, this microorganism consumes DHA produced in the culture medium. Here, we attempted to understand the pathway for DHA metabolism in NBRC3255 to minimize DHA degradation. The two gene products, NBRC3255_2003 (DhaK) and NBRC3255_3084 (DerK), have been annotated as DHA kinases in the NBRC 3255 draft genome. Because the double deletion derivative for dhaK and derK showed ATP-dependent DHA kinase activity similar to that of the wild type, we attempted to purify DHA kinase from ∆dhaK ∆derK cells to identify the gene for DHA kinase. The identified gene was NBRC3255_0651, of which the product was annotated as glycerol kinase (GlpK). Mutant strains with several combinations of deletions for the dhaK, derK, and glpK genes were constructed. The single deletion strain ∆glpK showed approximately 10% of wild-type activity and grew slower on glycerol than the wild type. The double deletion strain ∆derK ∆glpK and the triple deletion strain ∆dhaK ∆derK ∆glpK showed DHA kinase activity less than a detection limit and did not grow on glycerol. In addition, although ΔderK ΔglpK consumed a small amount of DHA in the late phase of growth, ∆dhaK ΔderK ΔglpK did not show DHA consumption on glucose-glycerol medium. The transformants of the ∆dhaK ΔderK ΔglpK strain that expresses one of the genes from plasmids showed DHA kinase activity. We concluded that all three DHA kinases, DhaK, DerK, and GlpK, are involved in DHA metabolism of G. thailandicus. KEY POINTS: • Dihydroxyacetone (DHA) is produced but degraded by Gluconobacter thailandicus. • Phosphorylation rather than reduction is the first committed step in DHA metabolism. • Three kinases are involved in DHA metabolism with the different properties.

Relocation of dehydroquinate dehydratase to the periplasmic space improves dehydroshikimate production with Gluconobacter oxydans strain NBRC3244.

3-Dehydroshikimate (3-DHS) is a key intermediate for the synthesis of various compounds, including the antiviral drug oseltamivir. The Gluconobacter oxydans strain NBRC3244 intrinsically oxidizes quinate to produce 3-dehydroquinate (3-DHQ) in the periplasmic space. Even though a considerable activity is detected in the recombinant G. oxydans homologously overexpressing type II dehydroquinate dehydratase (DHQase) encoded in the aroQ gene at a pH where it grows, an alkaline shift of the culture medium is required for 3-DHS production in the middle of cultivation. Here, we attempted to adopt type I DHQase encoded in the aroD gene of Gluconacetobacter diazotrophicus strain PAL5 because the type I DHQase works optimally at weak acid, which is preferable for growth conditions of G. oxydans. In addition, we anticipated that subcellular localization of DHQase is the cytoplasm, and therefore, transports of 3-DHQ and 3-DHS across the cytoplasmic membrane are rate-limiting steps in the biotransformation. The Sec- and TAT-dependent signal sequences for secretion were attached to the N terminus of AroD to change the subcellular localization. G. oxydans that expresses the TAT-AroD derivative achieved 3-DHS production at a tenfold higher rate than the reference strain that expresses wild-type AroD even devoid of alkaline shift. Enzyme activity with the intact cell suspension and signal sequence cleavage supported the relocation of AroD to the periplasmic space. The present study suggests that the relocation of DHQase improves 3-DHS production in G. oxydans and represents a proof of concept for the potential of enzyme relocation in metabolic engineering. KEY POINTS: • Type-I dehydroquinate dehydratase (DHQase) was expressed in Gluconobacter oxydans. • Cytoplasmic DHQase was relocated to the periplasmic space in G. oxydans. • Relocation of DHQase in G. oxydans improved 3-dehydroshikimate production.

Characterization of a cryptic, pyrroloquinoline quinone-dependent dehydrogenase of Gluconobacter sp. strain CHM43.

We characterized the pyrroloquinoline quinone (PQQ)-dependent dehydrogenase 9 (PQQ-DH9) of Gluconobacter sp. strain CHM43, which is a homolog of PQQ-dependent glycerol dehydrogenase (GLDH). We used a plasmid construct to express PQQ-DH9. The expression host was a derivative strain of CHM43, which lacked the genes for GLDH and the membrane-bound alcohol dehydrogenase and consequently had minimal ability to oxidize primary and secondary alcohols. The membranes of the transformant exhibited considerable d-arabitol dehydrogenase activity, whereas the reference strain did not, even if it had PQQ-DH9-encoding genes in the chromosome and harbored the empty vector. This suggests that PQQ-DH9 is not expressed in the genome. The activities of the membranes containing PQQ-DH9 and GLDH suggested that similar to GLDH, PQQ-DH9 oxidized a wide variety of secondary alcohols but had higher Michaelis constants than GLDH with regard to linear substrates such as glycerol. Cyclic substrates such as cis-1,2-cyclohexanediol were readily oxidized by PQQ-DH9.

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.

Comparative Genomic Analysis of Closely Related Acetobacter pasteurianus Strains Provides Evidence of Horizontal Gene Transfer and Reveals Factors Necessary for Thermotolerance.

Acetobacter pasteurianus is an industrial strain used for the vinegar production. Many A. pasteurianus strains with different phenotypic characteristics have been isolated so far. To understand the genetic background underpinning these phenotypes, a comparative genomic analysis of A. pasteurianus strains was conducted. Based on bioinformatics and experimental results, we report the following. (i) The gene repertoire related to the respiratory chains showed that several horizontal gene transfer events occurred after the divergence of these strains, indicating that the respiratory chain in A. pasteurianus has the diversity to adapt to its environment. (ii) There is a clear difference in thermotolerance even between 12 closely related strains. NBRC 3279, NBRC 3284, and NBRC 3283, in particular, which have only 55 mutations in total, showed differences in thermotolerance. The Na+/H+ antiporter gene nhaK2 was mutated in the thermosensitive NBRC 3279 and NBRC 3284 strains and not in the thermotolerant NBRC 3283 strain. The Na+/H+ antiporter activity of the three strains and expression of nhaK2 gene from NBRC 3283 in the two thermosensitive strains showed that these mutations are critical for thermotolerance. These results suggested that horizontal gene transfer events and several mutations have affected the phenotypes of these closely related strains.IMPORTANCEAcetobacter pasteurianus, an industrial vinegar-producing strain, exhibits diverse phenotypic differences such as respiratory activity related to acetic acid production, acetic acid resistance, or thermotolerance. In this study, we investigated the correlations between genome sequences and phenotypes among closely related A. pasteurianus strains. The gene repertoire related to the respiratory chains showed that the respiratory components of A. pasteurianus has a diversity caused by several horizontal gene transfers and mutations. In three closely related strains with clear differences in their thermotolerances, we found that the insertion or deletion that occurred in the Na+/H+ antiporter gene nhaK2 is directly related to their thermotolerance. Our study suggests that a relatively quick mutation has occurred in the closely related A. pasteurianus due to its genetic instability and that this has largely affected its phenotype.

Komagataeibacter diospyri sp. nov., a novel species of thermotolerant bacterial nanocellulose-producing bacterium.

Thermotolerant bacterial nanocellulose-producing strains, designated MSKU 9T and MSKU 15, were isolated from persimmon and sapodilla fruits, respectively. These strains were aerobic, Gram-stain-negative, had rod-shaped cells, were non-motile and formed white-cream colonies. Phylogeny based on the 16S rRNA gene sequences revealed that MSKU 9T and MSKU 15 represented members of the genus Komagataeibacter and formed a monophyletic branch with K. swingsii JCM 17123T and K. europaeus DSM 6160T. The genomic analysis revealed that overall genomic relatedness index values of MSKU 9T with K. swingsii JCM 17123T and K. europaeus DSM 6160T were ~90 % average nucleotide identity (ANI) and ≤58.2 % digital DNA-DNA hybridization (dDDH), respectively. MSKU 9T and MSKU 15 can be differentiated from the closely related K. swingsii JCM 17123T by their growth on 30 % d-glucose and ability to utilize and to form acid from raffinose and sucrose as carbon sources, and from K. europaeus DSM 6160T by their ability to grow without acetic acid. The genomic DNA G+C contents of MSKU 9T and MSKU 15 were 60.4 and 60.2 mol%, respectively. The major fatty acids of MSKU 9T and MSKU 15 were summed feature 8 (C18 : 1 ω7c and/or C18  : 1ω6c). The respiratory quinone was determined to be Q10. On the basis of the results of the polyphasic taxonomic analysis, MSKU 9T (=TBRC 9844T=NBRC 113802T) represents a novel species of the genus Komagataeibacter, for which the name Komagataeibacter diospyri sp. nov. is proposed.

Corynebacterium suranareeae sp. nov., a glutamate producing bacterium isolated from soil and its complete genome-based analysis.

Strain N24T was isolated from soil contaminated with starling's feces collected from Roi-Et province, Thailand. Cells of N24T were Gram-stain-positive rods, aerobic and non-spore-forming. N24T was positive for catalase, urease, citrate utilization, nitrate reduction and Methyl Red (MR) test but negative for oxidase, casein, gelatin liquefaction, tyrosine, Voges-Proskauer (VP) reaction and starch hydrolysis. Meso-diaminopimelic acid, rhamnose, ribose, arabinose and galactose were detected in its whole-cell hydrolysates. The results of the 16S rRNA gene sequence analysis indicated that N24T represented a member of the genus Corynebacterium. N24T was closely related to Corynebacterium glutamicum ATCC 13032T, with 99.0 % 16S rRNA gene sequence similarity. According to results obtained using in silico DNA-DNA hybridization approaches, N24T showed highest DNA-DNA relatedness (27.6 %) and average nucleotide identity (84.1 %) to Corynebacterium glutamicum ATCC 13032T. The DNA G+C content of N24T was 51.8 mol% (genome based). The major cellular fatty acids of N24T were C16 : 0, and C18 : 1ω9c. N24T had the nine isoprenes unit, MK-9(H2) as the predominant menaquinone. The predominant polar lipids were phosphatidylglycerol, phosphatidylinositol and diphosphatidylglycerol. Mycolic acids were also present. According to the complete genome sequence data, strain N24T and C. glutamicum ATCC 13032T are close phylogenetic neighbours, but have different genome characteristics. On the basis of the results of the genotypic and genomic studies and phenotypic characteristics including chemotaxonomy, strain N24T should be classified as representing a novel species of the genus Corynebacterium, for which the name Corynebacterium suranareeae sp. nov. is proposed. The type strain is N24T (TBRC 5845T=NBRC 113465T).

In vitro thermal adaptation of mesophilic Acetobacter pasteurianus NBRC 3283 generates thermotolerant strains with evolutionary trade-offs.

Thermotolerant strains are critical for low-cost high temperature fermentation. In this study, we carried out the thermal adaptation of A. pasteurianus IFO 3283-32 under acetic acid fermentation conditions using an experimental evolution approach from 37ºC to 40ºC. The adapted strain exhibited an increased growth and acetic acid fermentation ability at high temperatures, however, with the trade-off response of the opposite phenotype at low temperatures. Genome analysis followed by PCR sequencing showed that the most adapted strain had 11 mutations, a single 64-kb large deletion, and a single plasmid loss. Comparative phenotypic analysis showed that at least the large deletion (containing many ribosomal RNAs and tRNAs genes) and a mutation of DNA polymerase (one of the 11 mutations) critically contributed to this thermotolerance. The relationship between the phenotypic changes and the gene mutations are discussed, comparing with another thermally adapted A. pasteurianus strains obtained previously.