Naoya Kataoka

Development of butanol-tolerant Bacillus subtilis strain GRSW2-B1 as a potential bioproduction host

As alternative microbial hosts for butanol production with organic-solvent tolerant trait are in high demands, a butanol-tolerant bacterium, Bacillus subtilis GRSW2-B1, was thus isolated. Its tolerance covered a range of organic solvents at high concentration (5%v/v), with remarkable tolerance in particular to butanol and alcohol groups. It was susceptible for butanol acclimatization, which resulted in significant tolerance improvement. It has versatility for application in a variety of fermentation process because it has superior tolerance when cells were exposed to butanol either as high-density, late-exponential grown cells (up to 5%v/v) or under growing conditions (up to 2.25%v/v). Genetic transformation procedure was optimized, yielding the highest efficiency at 5.17 × 103 colony forming unit (μg DNA)-1. Gene expression could be effectively driven by several promoters with different levels, where as the highest expression was observed with a xylose promoter. The constructed vector was stably maintained in the transformants, in the presence or absence of butanol stress. Adverse effect of efflux-mediated tetracycline resistance determinant (TetL) to bacterial organic-solvent tolerance property was unexpectedly observed and thus discussed. Overall results indicate that B. subtilis GRSW2-B1 has potential to be engineered and further established as a genetic host for bioproduction of butanol.

Identification of CtpL as a chromosomally-encoded chemoreceptor for 4-chloroaniline and catechol in Pseudomonas aeruginosa PAO1

ABSTRACT Bacterial chemotaxis influences the ability of bacteria to survive and thrive in most environments, including polluted ones. Despite numerous reports of the phenotypic characterization of chemotactic bacteria, only a few molecular details of chemoreceptors for aromatic pollutants have been described. In this study, the molecular basis of chemotaxis toward an environmentally toxic chlorinated aromatic pollutant, 4-chloroaniline (4CA), was evaluated. Among the three Pseudomonas spp. tested, Pseudomonas aeruginosa PAO1 exhibited positive chemotaxis both to the nonmetabolizable 4CA, where 4-chloroacetanilide was formed as a dead-end transformation product, and to the metabolizable catechol. Molecular analysis of all 26 mutants with a disrupted methyl-accepting chemotaxis gene revealed that CtpL, a chromosomally encoded chemoreceptor, was responsible for the positive chemotactic response toward 4CA. Since CtpL has previously been described to be a major chemoreceptor for inorganic phosphate at low concentrations in PAO1, this report describes a fortuitous ability of CtpL to function toward aromatic pollutants. In addition, its regulation not only was dependent on the presence of the chemoattractant inducer but also was regulated by conditions of phosphate starvation. These results expand the range of known chemotactic transducers and their function in the environmental bacterium PAO1.

Efficient Production of 2,5-Diketo-d-Gluconate via Heterologous Expression of 2-Ketogluconate Dehydrogenase in Gluconobacter japonicus

ABSTRACT 2,5-Diketo-d-gluconate (2,5DKG) is a compound that can be the intermediate for d-tartrate and also vitamin C production. Although Gluconobacter oxydans NBRC3293 produces 2,5DKG from d-glucose via d-gluconate and 2-keto-d-gluconate (2KG), with accumulation of the product in the culture medium, the efficiency of 2,5DKG production is unsatisfactory because there is a large amount of residual d-gluconate at the end of the biotransformation process. Oxidation of 2KG to 2,5DKG is catalyzed by a membrane-bound flavoprotein-cytochrome c complex: 2-keto-gluconate dehydrogenase (2KGDH). Here, we studied the kgdSLC genes encoding 2KGDH in G. oxydans NBRC3293 to improve 2,5DKG production by Gluconobacter spp. The kgdS, kgdL, and kgdC genes correspond to the small, large, and cytochrome subunits of 2KGDH, respectively. The kgdSLC genes were cloned into a broad-host-range vector carrying a DNA fragment of the putative promoter region of the membrane-bound alcohol dehydrogenase gene of G. oxydans for expression in Gluconobacter spp. According to our results, 2KGDH that was purified from the recombinant Gluconobacter cells showed characteristics nearly the same as those reported previously. We also expressed the kgdSLC genes in a mutant strain of Gluconobacter japonicus NBRC3271 (formerly Gluconobacter dioxyacetonicus IFO3271) engineered to produce 2KG efficiently from a mixture of d-glucose and d-gluconate. This mutant strain consumed almost all of the starting materials (d-glucose and d-gluconate) to produce 2,5DKG quantitatively as a seemingly unique metabolite. To our knowledge, this is the first report of a Gluconobacter strain that produces 2,5DKG efficiently and homogeneously.

Enhancement of (R)-1,3-butanediol production by engineered Escherichia coli using a bioreactor system with strict regulation of overall oxygen transfer coefficient and pH

(R)-1,3-butanediol ((R)-1,3-BD) is an important substrate for the synthesis of industrial chemicals. Despite its large demand, a bioprocess for the efficient production of 1,3-BD from renewable resources has not been developed. We previously reported the construction of recombinant Escherichia coli that could efficiently produce (R)-1,3-BD from glucose. In this study, the fermentation conditions were optimized to further improve 1,3-BD production by the recombinant strain. A batch fermentation was performed with an optimized overall oxygen transfer coefficient (82.3 h−1) and pH (5.5); the 1,3-BD concentration reached 98.5 mM after 36 h with high-yield (0.444 mol (mol glucose)−1) and a high maximum production rate (3.63 mM h−1). In addition, a fed-batch fermentation enabled the recombinant strain to produce 174.8 mM 1,3-BD after 96 h cultivation with a yield of 0.372 mol (mol glucose)−1, a maximum production rate of 3.90 mM h−1, and a 98.6% enantiomeric excess (% ee) of (R)-1,3-BD. Graphical Abstract E. coli engineered to synthesize (R)-1,3-butanediol from glucose provides the possibility to produce industrial chemicals from renewable resources.

Construction of CoA-dependent 1-butanol synthetic pathway functions under aerobic conditions in Escherichia coli

1-Butanol is an important industrial platform chemical and an advanced biofuel. While various groups have attempted to construct synthetic pathways for 1-butanol production, efforts to construct a pathway that functions under aerobic conditions have met with limited success. Here, we constructed a CoA-dependent 1-butanol synthetic pathway that functions under aerobic conditions in Escherichia coli, by expanding the previously reported (R)-1,3-butanediol synthetic pathway. The pathway consists of phaA (acetyltransferase) and phaB (NADPH-dependent acetoacetyl-CoA reductase) from Ralstonia eutropha, phaJ ((R)-specific enoyl-CoA hydratase) from Aeromonas caviae, ter (trans-enoyl-CoA reductase) from Treponema denticola, bld (butylraldehyde dehydrogenase) from Clostridium saccharoperbutylacetonicum, and inherent alcohol dehydrogenase(s) from E. coli. To evaluate the potential of this pathway for 1-butanol production, culture conditions, including volumetric oxygen transfer coefficient (kLa) and pH were optimized in a mini-jar fermenter. Under optimal conditions, 1-butanol was produced at a concentration of up to 8.60gL(-1) after 46h of fed-batch cultivation.

Membrane-bound glycerol dehydrogenase catalyzes oxidation of D-pentonates to 4-keto-D-pentonates, D-fructose to 5-keto-D-fructose, and D-psicose to 5-keto-D-psicose

A novel oxidation of D-pentonates to 4-keto-D-pentonates was analyzed with Gluconobacter thailandicus NBRC 3258. D-Pentonate 4-dehydrogenase activity in the membrane fraction was readily inactivated by EDTA and it was reactivated by the addition of PQQ and Ca2+. D-Pentonate 4-dehydrogenase was purified to two different subunits, 80 and 14 kDa. The absorption spectrum of the purified enzyme showed no typical absorbance over the visible regions. The enzyme oxidized D-pentonates to 4-keto-D-pentonates at the optimum pH of 4.0. In addition, the enzyme oxidized D-fructose to 5-keto-D-fructose, D-psicose to 5-keto-D-psicose, including the other polyols such as, glycerol, D-ribitol, D-arabitol, and D-sorbitol. Thus, D-pentonate 4-dehydrogenase was found to be identical with glycerol dehydrogenase (GLDH), a major polyol dehydrogenase in Gluconobacter species. The reaction versatility of quinoprotein GLDH was notified in this study.

A novel Na+(K+)/H+ antiporter plays an important role in the growth of Acetobacter tropicalis SKU1100 at high temperatures via regulation of cation and pH homeostasis

A gene encoding a putative Na(+)/H(+) antiporter was previously proposed to be involved in the thermotolerance mechanism of Acetobacter tropicalis SKU 1100. The results of this study show that disruption of this antiporter gene impaired growth at high temperatures with an external pH>6.5. The growth impairment at high temperatures was much more severe in the absence of Na(+) (with only the presence of K(+)); under these conditions, cells failed to grow even at 30°C and neutral to alkaline pH values, suggesting that this protein is also important for K(+) tolerance. Functional analysis with inside-out membrane vesicles from wild type and mutant strains indicated that the antiporter, At-NhaK2 operates as an alkali cation/proton antiporter for ions such as Na(+), K(+), Li(+), and Rb(+) at acidic to neutral pH values (6.5-7.5). The membrane vesicles were also shown to contain a distinct pH-dependent Na(+)(specific)/H(+) antiporter(s) that might function at alkaline pH values. In addition, phylogenetic analysis showed that At-NhaK2 is a novel type of Na(+)/H(+) antiporter belonging to a phylogenetically distinct new clade. These data demonstrate that At-NhaK2 functions as a Na(+)(K(+))/H(+) antiporter and is essential for K(+) and pH homeostasis during the growth of A. tropicalis SKU1100, especially at higher temperatures.

A Single-Nucleotide Insertion in a Drug Transporter Gene Induces a Thermotolerance Phenotype in Gluconobacter frateurii by Increasing the NADPH/NADP+ Ratio via Metabolic Change

ABSTRACT Thermotolerant microorganisms are beneficial to the fermentation industry because they reduce the need for cooling and offer other operational advantages. Previously, we obtained a thermally adapted Gluconobacter frateurii strain by experimental evolution. In the present study, we found only a single G insertion in the adapted strain, which causes a frameshift in a gene encoding a putative drug transporter. A mutant derivative strain with the single G insertion in the transporter gene (Wild-G) was constructed from the wild-type strain and showed increased thermotolerance. We found that the thermotolerant strains accumulated substantial intracellular trehalose and manifested a defect in sorbose assimilation, suggesting that the transporter is partly involved in trehalose efflux and sorbose uptake and that the defect in the transporter can improve thermotolerance. The ΔotsAB strain, constructed by elimination of the trehalose synthesis gene in the wild type, showed no trehalose production but, unexpectedly, much better growth than the adapted strain at high temperatures. The ΔotsAB mutant produced more acetate as the final metabolite than the wild-type strain did. We hypothesized that trehalose does not contribute to thermotolerance directly; rather, a metabolic change including increased carbon flux to the pentose phosphate pathway may be the key factor. The NADPH/NADP+ ratio was higher in strain Wild-G, and much higher in the ΔotsAB strain, than in the wild-type strain. Levels of reactive oxygen species (ROS) were lower in the thermotolerant strains. We propose that the defect of the transporter causes the metabolic flux to generate more NADPH, which may enhance thermotolerance in G. frateurii. IMPORTANCE The biorefinery industry has to ensure that microorganisms are robust and retain their viability and function at high temperatures. Here we show that Gluconobacter frateurii, an industrially important member of the acetic acid bacteria, exhibited enhanced thermotolerance through the reduction of trehalose excretion after thermal adaptation. Although intracellular trehalose may play a key role in thermotolerance, the molecular mechanisms of action of trehalose in thermotolerance are a matter of debate. Our mutated strain that was defective in trehalose synthase genes, producing no trehalose but a larger amount of acetic acid as the end metabolite instead, unexpectedly showed higher thermotolerance than the wild type. Our adapted and mutated thermotolerant strains showed increased NADPH/NADP+ ratios and reductions in ROS levels. We concluded that in G. frateurii, trehalose does not contribute to thermotolerance directly; rather, the metabolic change increases the NADPH/NADP+ ratio to enhance thermotolerance.

Aldopentoses as new substrates for the membrane-bound, pyrroloquinoline quinone-dependent glycerol (polyol) dehydrogenase of Gluconobacter sp.

Membrane-bound, pyrroloquinoline quinone (PQQ)-dependent glycerol dehydrogenase (GLDH, or polyol dehydrogenase) of Gluconobacter sp. oxidizes various secondary alcohols to produce the corresponding ketones, such as oxidation of D-sorbitol to L-sorbose in vitamin C production. Substrate specificity of GLDH is considered limited to secondary alcohols in the D-erythro configuration at the next to the last carbon. Here, we suggest that L-ribose, D- and L-lyxoses, and L-tagatose are also substrates of GLDH, but these sugars do not meet the substrate specificity rule of GLDH. The oxygen consumption activity of wild-type Gluconobacter frateurii cell membranes depends on several kinds of sugars as compared with that of the membranes of a GLDH-negative variant. Biotransformation of those sugars with the membranes was examined to determine the reaction products. A time course measuring the pH in the reaction mixture and the increase or decrease in substrates and products on TLC suggested that oxidation products of L-lyxose and L-tagatose were ketones with unknown structures, but those of L-ribose and D-lyxose were acids. The oxidation product of L-ribose was purified and revealed to be L-ribonate by HRMS and NMR analysis. Biotransformation of L-ribose with the membranes and also with the whole cells produced L-ribonate in nearly stoichiometric amounts, indicating that the specific oxidation site in L-ribose is recognized by GLDH. Since purified GLDH produced L-ribonate without any intermediate-like compounds, we propose here a reaction model where the first carbon in the pyranose form of L-ribose is oxidized by GLDH to L-ribonolactone, which is further hydrolyzed spontaneously to produce L-ribonate.

Role of a membrane-bound aldehyde dehydrogenase complex AldFGH in acetic acid fermentation with Acetobacter pasteurianus SKU1108

Acetic acid fermentation is widely considered a consequence of ethanol oxidation by two membrane-bound enzymes—alcohol dehydrogenase and aldehyde dehydrogenase (ALDH)—of acetic acid bacteria. Here, we used a markerless gene disruption method to construct a mutant of the Acetobacter pasteurianus strain SKU1108 with a deletion in the aldH gene, which encodes the large catalytic subunit of a heterotrimeric ALDH complex (AldFGH), to examine the role of AldFGH in acetic acid fermentation. The ΔaldH strain grew less on ethanol-containing medium, i.e., acetic acid fermentation conditions, than the wild-type strain and significantly accumulated acetaldehyde in the culture medium. Unexpectedly, acetaldehyde oxidase activity levels of the intact ΔaldH cells and the ΔaldH cell membranes were similar to those of the wild-type strain, which might be attributed to an additional ALDH isozyme (AldSLC). The apparent KM values of the wild-type and ΔaldH membranes for acetaldehyde were similar to each other, when the cells were cultured in nonfermentation conditions, where ΔaldH cells grow as well as the wild-type cells. However, the membranes of the wild-type cells grown under fermentation conditions showed a 10-fold lower apparent KM value than those of the cells grown under nonfermentation conditions. Under fermentation conditions, transcriptional levels of a gene for AldSLC were 10-fold lower than those under nonfermentation conditions, whereas aldH transcript levels were not dramatically changed under the two conditions. We suggest that A. pasteurianus SKU1108 has two ALDHs, and the AldFGH complex is indispensable for acetic acid fermentation and is the major enzyme under fermentation conditions.