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Microbiologically influenced corrosion (MIC) is caused by biofilms formed on metal surfaces, and MIC of metal alloys on marine infrastructure leads to severe accidents and great economic losses. Although bacterial community analyses of the biofilms collected from corroded metal have been studied, the analyses of biofilms collected from uncorroded metal are rarely reported. In this study, a biofilm formed on an uncorroded metal joint attached to a metal dock mooring at Akitsu Port was used as a model for bacterial community analysis. The bacterial community was analyzed by high-throughput sequencing of the V3–V4 variable regions of the 16S rRNA gene. Bacterial species contained in the biofilms were identified at the genus level, and Alkanindiges bacteria were the dominant species, which have been not reported as the dominant species in previous research on MIC. The genome sequences of known Alkanindiges bacteria do not have conserved gene clusters required to cause metal corrosion, which suggests that Alkanindiges bacteria do not corrode metals but act on the formation of biofilms. Those findings indicated that the bacterial community may change significantly during the process from biofilm formation to the occurrence of metal corrosion.

Metagenomic MeBglD2 is a glycoside hydrolase family 1 (GH1) β-glycosidase that has β-glucosidase, β-fucosidase, and β-galactosidase activities, and is highly activated in the presence of monosaccharides and disaccharides. The β-glucosidase activity of MeBglD2 increases in a cellobiose concentration-dependent manner and is not inhibited by a high concentration of D-glucose or cellobiose. Previously, we solved the crystal structure of MeBglD2 and designed a thermostable mutant; however, the mechanism of substrate recognition of MeBglD2 remains poorly understood. In this paper, we report the X-ray crystal structures of MeBglD2 complexed with various saccharides, such as D-glucose, D-xylose, cellobiose, and maltose. The results showed that subsite - 1 of MeBglD2, which contained two catalytic glutamate residues (a nucleophilic Glu356 and an acid/base Glu170) was common to other GH1 enzymes, but the positive subsites (+ 1 and + 2) had different binding modes depending on the type of sugar. Three residues (Glu183, Asn227, and Asn229), located at the positive subsites of MeBglD2, were involved in substrate specificity toward cellobiose and/or chromogenic substrates in the presence of additive sugars. The docking simulation of MeBglD2-cellobiose indicated that Asn229 and Trp329 play important roles in the recognition of + 1 D-glucose in cellobiose. Our findings provide insights into the unique substrate recognition mechanism of GH1, which can incorporate a variety of saccharides into its positive subsites. KEY POINTS: • Metagenomic glycosidase, MeBglD2, recognizes various saccharides • Structures of metagenomic MeBglD2 complexed with various saccharides are determined • MeBglD2 has a unique substrate recognition mechanism at the positive subsites.

Aspergillus oryzae isoprimeverose-producing oligoxyloglucan hydrolase (IpeA) releases isoprimeverose units (α-d-xylopyranosyl-(1→6)-d-glucose) from the non-reducing end of xyloglucan oligosaccharides and belongs to glycoside hydrolase family 3. In this paper, we report the X-ray crystal structure of the IpeA complexed with a xyloglucan oligosaccharide, (XXXG: Glc

Strain CCI5, an oligotrophic bacterium, was isolated from leaf soil collected in Japan. Strain CCI5 grew at temperatures between 25 °C and 43 °C (optimum temperature, 40 °C) and at pHs between 6.0 and 10.0 (optimum pH, 9.0). Its major fatty acids were anteiso-C15:0 and iso-C16:0, and menaquinone 7 was the only detected quinone system. In a phylogenetic analysis based on 16S rRNA gene sequences, strain CCI5 presented as a member of the genus Paenibacillus. Moreover, multilocus sequence analysis based on partial sequences of the atpD, dnaA, gmk, and infB genes showed that strain CCI5 tightly clustered with P. glycanilyticus DS-1T. The draft genome of strain CCI5 consisted of 6,864,972 bp with a G+C content of 50.7% and comprised 6,189 predicted coding sequences. The genome average nucleotide identity value (97.8%) between strain CCI5 and P. glycanilyticus DS-1T was below the cut-off value for prokaryotic subspecies delineation. Based on its phenotypic, chemotaxonomic, and phylogenetic features, strain CCI5 (= HUT-8145T = KCTC 43270T) can be considered as a novel subspecies within the genus Paenibacillus with the proposed name Paenibacillus glycanilyticus subsp. hiroshimensis subsp. nov.

Strain AS-1 was isolated from laboratory-scale activated sludge collected in Japan. This strain not only grows on rich medium, including R2A medium, but also forms colonies on medium lacking organic matter other than agar (water agar), indicating it could be used as a eurytrophic recombinant host in material production processes. Here, we present a draft genome sequence of Enterobacter sp. AS-1, which consists of a total of 24 contigs containing 5,207,146 bp, with a GC content of 55.64%, and comprising 4,921 predicted coding sequences. Based on 16S rRNA gene sequence analysis, strain AS-1 was designated as Enterobacter sp. AS-1.

Strain CCI9, which was isolated from leaf soil collected in Japan, was capable of growth on poor-nutrient medium, at temperatures of 10°C to 45°C, at pHs of 4.5 to 10, and in the presence of 7.0% NaCl. We determined a draft genome sequence of strain CCI9, which consists of a total of 28 contigs containing 4,644,734 bp with a GC content of 56.1%. This assembly yielded 4,154 predicted coding sequences. Multilocus sequence analysis (MLSA) based on atpD, gyrB, infB, and rpoB gene sequences were performed to further identify strain CCI9. The MLSA revealed that strain CCI9 clustered tightly with Enterobacter roggenkampii EN-117T. Moreover, the average nucleotide identity value (98.6%) between genome sequences of strain CCI9 and E. roggenkampii EN-117T exceeds the cutoff value for prokaryotic subspecies delineation. Therefore, strain CCI9 was identified as E. roggenkampii CCI9. To clarify differences between E. roggenkampii EN-117T and CCI9, the coding proteins were compared against the eggNOG database.

An aerobic, oligotrophic, Gram-positive, non-sporulating, motile, rod-shaped, palladium-leaching bacterial strain, Deinococcus sp. KR-1, was previously isolated from pond water collected in Japan. This strain grew at 10 °C to 40 °C (optimum 30 °C), at pH 4.5 to 11.5 (optimum pH 8.0), and in the presence of 2.0% NaCl. Its major cellular fatty acids were C15: 1ω6 and C16 : 1ω7c. The quinone system was menaquinone 8. Multilocus sequence analysis based on partial sequences of four housekeeping genes (atpD, dnaA, gyrB and rpoB) showed that branching of Deinococcus sp. KR-1 was distant to those of Deinococcus type strains. The genome average nucleotide identity value between strain KR-1 and its closest related Deinococcus type strain was less than 95.69%. Based on its phenotypic, chemotaxonomic and phylogenetic data, strain KR-1 (= HUT-8138T = KCTC 33977T) can be considered a novel species within the genus Deinococcus with the proposed name Deinococcus kurensis sp. nov.

The discharge of industrial dyes and their breakdown products are often environmentally harmful. Here, we describe a biodegradation method using Burkholderia multivorans CCA53, which exhibits a capacity to degrade azo dyes, particularly ethyl red. Under the optimized culture conditions, 100 μM ethyl red was degraded more than 99% after incubation for 8 h. Real-time PCR analysis of azoR1 and azoR2, encoding two azoreductases, revealed that transcription level of these genes is enhanced at early phase under the optimized conditions. For a more practical approach, hydrolysates were prepared from eucalyptus or Japanese cedar chips or rice straw, and rice straw hydrolysate was used as the best medium for ethyl red biodegradation. Under those conditions, ethyl red was also degraded with high efficiency (>91%). We have thus constructed a potentially economical method for the biodegradation of ethyl red.

Construction of acid-tolerant strains of Saccharomyces cerevisiae is required for various bioproduction processes. We previously isolated the gene IoGAS1 from multiple stress-tolerant Issatchenkia orientalis as a gene conferring sulfuric acid resistance in S. cerevisiae, but its acid tolerance was only investigated using sulfuric acid. Here, we evaluated the growth and ethanol fermentation ability of the IoGAS1-expressing S. cerevisiae strain, B4-IoGAS1, by using various acidic reagents. B4-IoGAS1 exhibited faster growth than the control strain, B4-CON, when cultured aerobically with sulfuric, hydrochloric, formic, acetic, and lactic acids at pH below 2.4. However, the growth of B4-IoGAS1 was suppressed at pH above 2.48, irrespective of the type of acid reagents. Furthermore, B4-IoGAS1 exhibited higher performance of ethanol fermentation than B4-CON under 250 mM lactic acid condition at pH 2.37. These results demonstrate that IoGAS1 could facilitate the aerobic growth and anaerobic ethanol production under different acidic stressed conditions.

Klebsiella pneumoniae subsp. pneumoniae CCI2 was isolated from leaf soil collected in Hiroshima Prefecture, Japan. The draft genome sequence comprises 78 contigs and contains 5,075,115 bp with a G+C content of 57.7%.

Klebsiella pneumoniae subsp. pneumoniae CCI2 was isolated from leaf soil collected in Hiroshima Prefecture, Japan. The draft genome sequence comprises 78 contigs and contains 5,075,115 bp with a G+C content of 57.7%.

Enterobacter oligotrophicus CCA3 was isolated from leaf soil collected in Hiroshima Prefecture, Japan. Here, we report the draft genome sequence of E. oligotrophicus CCA3. The draft genome sequence of E. oligotrophicus CCA3 consists of 29 contigs of 4,425,100 bp, with a GC content of 54.2%.

Strain CCI5, an oligotrophic bacterium, was isolated from leaf soil collected in Japan. Strain CCI5 grew at temperatures between 25 °C and 43 °C (optimum temperature, 40 °C) and at pHs between 6.0 and 10.0 (optimum pH, 9.0). Its major fatty acids were anteiso-C

meso‐Diaminopimelate dehydrogenase (meso‐DAPDH) catalyzes the reversible NADP+‐dependent oxidative deamination of meso‐2,6‐diaminopimelate (meso‐DAP) to produce l‐2‐amino‐6‐oxopimelate. Moreover, d‐amino acid dehydrogenase (d‐AADHs) derived from protein‐engineered meso‐DAPDH is useful for one‐step synthesis of d‐amino acids with high optical purity. Here, we report the identification and functional characterization of a novel NAD(P)+‐dependent meso‐DAPDH from Numidum massiliense (NmDAPDH). After the gene encoding the putative NmDAPDH was expressed in recombinant Escherichia coli cells, the enzyme was purified 4.0‐fold to homogeneity from the crude extract through five purification steps. Although the previously known meso‐DAPDHs use only NADP+ as a coenzyme, NmDAPDH was able to use both NADP+ and NAD+ as coenzymes. When NADP+ was used as a coenzyme, NmDAPDH exhibited an approximately 2 times higher kcat/Km value toward meso‐DAP than that of meso‐DAPDH from Symbiobacterium thermophilum (StDAPDH). NmDAPDH also catalyzed the reductive amination of corresponding 2‐oxo acids to produce acidic d‐amino acids such as d‐aspartate and d‐glutamate. The optimum pH and temperature for the oxidative deamination of meso‐DAP were about 10.5 and 75°C, respectively. Like StDAPDH, NmDAPDH exhibited high stability: it retained more than 75% of its activity after 30 min at 60°C (pH 7.2) or at pHs ranging from 5.5 to 13.0 (50°C). Alignment of the amino acid sequences of NmDAPDH and the known meso‐DAPDHs suggested NmDAPDH has a hexameric structure. Given its specificity for both NADP+ and NAD+, high stability, and a broad range of reductive amination activity toward 2‐oxo acids, NmDAPDH appears to offer advantages for engineering a more effective d‐AADH.

Pyruvate, a potential precursor of various chemicals, is one of the fundamental chemicals produced by the fermentation process. We previously reported a pyruvate-producing Escherichia coli strain LAFCPCPt-accBC-aceE (PYR) that has the potential to be applied to the industrial production of pyruvate. In this study, the availability of the PYR strain for the production of pyruvate-derivative chemicals was evaluated using a d-lactate-producing strain (LAC) based on the PYR strain. The LAC strain expresses a d-lactate dehydrogenase-encoding gene from Lactobacillus bulgaricus under the control of a T7 expression system. The d-lactate productivity of the LAC strain was further improved by limiting aeration and changing the induction period for the expression of d-lactate dehydrogenase-encoding gene expression. Under combined conditions, the LAC strain produced d-lactate at 21.7 &plusmn, 1.4 g&middot, L&minus, 1, which was compatible with the pyruvate production by the PYR strain (26.1 &plusmn, 0.9 g&middot, 1). These results suggest that we have succeeded in the effective conversion of pyruvate to d-lactate in the LAC strain, demonstrating the wide versatility of the parental PYR strain as basal strain for various chemicals production.

meso-Diaminopimelate dehydrogenase (meso-DAPDH) catalyzes the reversible NADP

To generate an organism capable of producing d-lactate, NAD+-dependent d-lactate dehydrogenase was expressed in our pyruvate-producing strain, Escherichia coli strain LAFCPCPt-accBC-aceE. After determining the optimal culture conditions for d-lactate production, 18.4 mM d-lactate was produced from biomass-based medium without supplemental mineral or nitrogen sources. Our results show that d-lactate can be produced in simple batch fermentation processes.

Strain KR-1 was isolated from pond water collected in Japan. Because this strain was capable of adsorbing palladium particles in sterilized water, strain KR-1 will be a useful biocatalyst for palladium-leaching from metal waste. Here we present a draft genome sequence of Deinococcus sp. KR-1, which consists of a total of 7 contigs containing 4,556,772 bp with a GC content of 70.0% and comprises 4,450 predicted coding sequences. Based on the 16S rRNA gene sequence analysis, strain KR-1 was identified as Deinococcus sp. KR-1.

We previously reported the crystal structure of an acetyl esterase (TcAE206) belonging to carbohydrate esterase family 3 from Talaromyces cellulolyticus. In this study, we solved the crystal structure of an S10A mutant of TcAE206 complexed with an acetate ion. The acetate ion was stabilized by three hydrogen bonds in the oxyanion hole instead of a water molecule as in the structure of wild-type TcAE206. Furthermore, the catalytic triad residue His182 moved 0.8 Å toward the acetate ion upon substrate entering the active site, suggesting that this movement is necessary for completion of the catalytic reaction., Highlights • The crystal structure of TcAE206_S10A with acetate ion was solved at 1.4 Å resolution. • The complex structure revealed the catalytic mechanism. • His182, which moves 0.8 Å toward the acetate ion, is necessary for enzymatic activity. • This study provides insights into the substrate specificity of fungal CE3 enzymes.

meso-Diaminopimelate dehydrogenase (meso-DAPDH) catalyzes the reversible NADP+-dependent oxidative deamination of meso-2,6-diaminopimelate (meso-DAP) to produce l -2-amino-6-oxopimelate. meso-DAPDH is divided into two major clusters, types I and II, based on substrate specificity and structural characteristic. Here, we describe a novel type II meso-DAPDH from Thermosyntropha lipolytica (TlDAPDH). The gene encoding a putative TlDAPDH was expressed in Escherichia coli cells, and then the enzyme was purified 7.3-fold to homogeneity from the crude cell extract. The molecule of TlDAPDH seemed to form a hexamer, which is the typical structural characteristic of type II meso-DAPDHs. The purified enzyme exhibited oxidative deamination activity toward meso-DAP with both NADP+ and NAD+ as coenzymes. TlDAPDH exhibited reductive amination activity of corresponding 2-oxo acid to produce d -amino acid. In particular, the productivities for d -aspartate and d -glutamate have not been reported in the type II enzymes. The optimum pH and temperature for oxidative deamination of meso-DAP were 10.5 and 55°C, respectively. TlDAPDH retained more than 80% of its activity after incubation for 30 min at temperatures between 50°C and 65°C and in the pH range of 4.5–9.5. Moreover, the coenzyme and substrate recognition mechanisms of TlDAPDH were elucidated based on a multiple sequence alignment and the homology model. The results of these analyses suggested that the molecular mechanisms for coenzyme and substrate recognition of TlDAPDH were similar to those of meso-DAPDH from S. thermophilum, which is the representative type II enzyme. Based on the kinetic characteristics and structural comparison, TlDAPDH was considered to be a novel type II meso-DAPDH.

An aerobic, Gram-negative, non-sporulating, motile, rod-shaped and lignin-degrading bacterial strain, Pseudomonas sp. CCA1, was isolated from leaf soil collected in Japan. This strain grew at 20–45 °C (optimum 20 °C), at pH 5.0–10.0 (optimum pH 5.0), and in the presence of 2% NaCl. Its major cellular fatty acids were C16:0 and summed feature 8 (C18:1ω6c and/or C18:1ω7c). The predominant quinone system was ubiquinone-9. Comparative 16S rRNA gene sequence analysis showed that Pseudomonas sp. CCA1 was related most closely to P. citronellolis NBRC 103043T (98.9%), but multilocus sequence analysis based on fragments of the atpD, gyrA, gyrB and rpoB gene sequences showed strain CCA1 to branch separately from its most closely related Pseudomonas type strains. DNA–DNA hybridization values between Pseudomonas sp. CCA1 and type strains of closely related Pseudomonas species were less than 53%. Based on its phenotypic, chemotaxonomic and phylogenetic features, we propose that Pseudomonas sp. CCA1 represents a novel species within the genus Pseudomonas, for which the name Pseudomonas humi sp. nov. is proposed. The type strain is CCA1 (= HUT 8136T = TBRC 8616T).

Isobutanol is attracting attention as a potential biofuel because it has higher energy density and lower hygroscopicity than ethanol. To date, several effective methods for microbial production of isobutanol have been developed, but they require expensive reagents to maintain expression plasmids and induce expression, which is not suitable for practical production. Here, we describe a simple and efficient method for isobutanol production in Escherichia coli. It is noteworthy that no expression plasmids or inducers were used during the production. Instead, heterologous genes necessary for isobutanol production were all knocked into the genome, and the expression of those genes was induced by xylose, which is present in most biomass feedstocks. The constructed strain (mlcXT7-LAFC-AAKCD) contains Bacillus subtilis alsS, E. coli ilvCD, Lactococcus lactis adhA, and L. lactis kivd genes in its genome and efficiently produced isobutanol from glucose and xylose in flask batch cultures. Under conditions in which the temperature and pH of the medium and the aeration in the culture were all optimized, the final isobutanol concentration reached 8.4 g L(-1) after 48 h. Isobutanol was also produced using hydrolysate from Japanese cedar as the carbon source without supplemented glucose, xylose, or yeast extract. Under those conditions, isobutanol (3.7 g L(-1)) was produced in 96 h. Taken together, these results indicate that the developed strain is potentially useful for industrial isobutanol production.

Ureibacillus thermosphaericus A1 was isolated from compost collected in Munakata City, Fukuoka Prefecture, Japan. Here, we report the first complete genome sequence of U. thermosphaericus. The complete genome of this strain consists of 3,488,104 bp with a GC content of 36.3% and comprises 3,362 predicted coding sequences.

Ureibacillus thermosphaericus A1 was isolated from compost collected in Munakata City, Fukuoka Prefecture, Japan. Here, we report the first complete genome sequence of U. thermosphaericus . The complete genome of this strain consists of 3,488,104 bp with a GC content of 36.3% and comprises 3,362 predicted coding sequences.

Acetoin is used in the biochemical, chemical and pharmaceutical industries. Several effective methods for acetoin production from petroleum-based substrates have been developed, but they all have an environmental impact and do not meet sustainability criteria. Here we describe a simple and efficient method for acetoin production from oil palm mesocarp fiber hydrolysate using engineered Escherichia coli. An optimization of culture conditions for acetoin production was carried out using reagent-grade chemicals. The final concentration reached 29.9gL-1 with a theoretical yield of 79%. The optimal pretreatment conditions for preparing hydrolysate with higher sugar yields were then determined. When acetoin was produced using hydrolysate fortified with yeast extract, the theoretical yield reached 97% with an acetoin concentration of 15.5gL-1. The acetoin productivity was 10-fold higher than that obtained using reagent-grade sugars. This approach makes use of a compromise strategy for effective utilization of oil palm biomass towards industrial application.

A stable NADP + -dependent d -amino acid dehydrogenase (DAADH) was recently created from Ureibacillus thermosphaericus meso -diaminopimelate dehydrogenase through site-directed mutagenesis. To produce a novel DAADH mutant with different substrate specificity, the crystal structure of apo-DAADH was determined at a resolution of 1.78 Å, and the amino acid residues responsible for the substrate specificity were evaluated using additional site-directed mutagenesis. By introducing a single D94A mutation, the enzyme's substrate specificity was dramatically altered; the mutant utilized d -phenylalanine as the most preferable substrate for oxidative deamination and had a specific activity of 5.33 μmol/min/mg at 50°C, which was 54-fold higher than that of the parent DAADH. In addition, the specific activities of the mutant toward d -leucine, d -norleucine, d -methionine, d -isoleucine, and d -tryptophan were much higher (6 to 25 times) than those of the parent enzyme. For reductive amination, the D94A mutant exhibited extremely high specific activity with phenylpyruvate (16.1 μmol/min/mg at 50°C). The structures of the D94A-Y224F double mutant in complex with NADP + and in complex with both NADPH and 2-keto-6-aminocapronic acid (lysine oxo-analogue) were then determined at resolutions of 1.59 Å and 1.74 Å, respectively. The phenylpyruvate-binding model suggests that the D94A mutation prevents the substrate phenyl group from sterically clashing with the side chain of Asp94. A structural comparison suggests that both the enlarged substrate-binding pocket and enhanced hydrophobicity of the pocket are mainly responsible for the high reactivity of the D94A mutant toward the hydrophobic d -amino acids with bulky side chains. IMPORTANCE In recent years, the potential uses for d -amino acids as source materials for the industrial production of medicines, seasonings, and agrochemicals have been growing. To date, several methods have been used for the production of d -amino acids, but all include tedious steps. The use of NAD(P) + -dependent d -amino acid dehydrogenase (DAADH) makes single-step production of d -amino acids from oxo-acid analogs and ammonia possible. We recently succeeded in creating a stable DAADH and demonstrated that it is applicable for one-step synthesis of d -amino acids, such as d -leucine and d -isoleucine. As the next step, the creation of an enzyme exhibiting different substrate specificity and higher catalytic efficiency is a key to the further development of d -amino acid production. In this study, we succeeded in creating a novel mutant exhibiting extremely high catalytic activity for phenylpyruvate amination. Structural insight into the mutant will be useful for further improvement of DAADHs.

In this study, we describe a novel method for producing valuable chemicals from glucose and xylose in Escherichia coli. The notable features in our method are avoidance of plasmids and expensive inducers for foreign gene expression to reduce production costs; foreign genes are knocked into the chromosome, and their expression is induced with xylose that is present in most biomass feedstock. As loci for the gene knock-in, lacZYA and some pseudogenes are chosen to minimize unexpected effects of the knock-in on cell physiology. The promoter of xylF is inducible with xylose and is combined with the T7 RNA polymerase-T7 promoter system to ensure strong gene expression. This expression system was named BICES (biomass-inducible chromosome-based expression system). As examples of BICES application, 2,3-butanediol and acetoin were successfully produced from glucose and xylose, and the maximal concentrations reached 54gL(-1) [99.6% in (R,S)-form] and 31gL(-1), respectively. 2,3-Butanediol and acetoin are industrially important chemicals that are, at present, produced primarily through petrochemical processes. To demonstrate usability of BICES in practical situations, we produced these chemicals from a saccharified cedar solution. From these results, we can conclude that BICES is suitable for practical production of valuable chemicals from biomass.

D-isoleucine (D-Ile) can be assayed using chiral chromatography but the availability of that method is limited by the necessity for special expertise and expensive equipment. We therefore developed a simple and specific colorimetric assay system for D-Ile determination using an artificially created NADP(+)-dependent D-amino acid dehydrogenase (DAADH). The system consists of two reaction steps: the first is the quantitative conversion of D-Ile to (3R)-2-oxo-3-methyl valerate by DAADH in which NADP(+) is converted to NADPH, while the second is chemical conversion of NADPH to reduced water-soluble Tetrazolium-3 via a redox mediator. D-Ile was determined from 1 to 50 µM, and the assay was unaffected by the presence of any of three other isomers (100 µM), alcohol and organic acids.

D-Branched-chain amino acids (D-BCAAs) such as D-leucine, D-isoleucine, and D-valine are known to be peptide antibiotic intermediates and to exhibit a variety of bioactivities. Consequently, much effort is going into achieving simple stereospecific synthesis of D-BCAAs, especially analogs labeled with stable isotopes. Up to now, however, no effective method has been reported. Here, we report the establishment of an efficient system for enantioselective synthesis of D-BCAAs and production of D-BCAAs labeled with stable isotopes. This system is based on two thermostable enzymes: D-amino acid dehydrogenase, catalyzing NADPH-dependent enantioselective amination of 2-oxo acids to produce the corresponding D-amino acids, and glucose dehydrogenase, catalyzing NADPH regeneration from NADP(+) and D-glucose. After incubation with the enzymes for 2 h at 65°C and pH 10.5, 2-oxo-4-methylvaleric acid was converted to D-leucine with an excellent yield (99 %) and optical purity (99 %). Using this system, we produced five different D-BCAAs labeled with stable isotopes: D-[1-(13)C,(15)N]leucine, D-[1-(13)C]leucine, D-[(15)N]leucine, D-[(15)N]isoleucine, and D-[(15)N]valine. The structure of each labeled D-amino acid was confirmed using time-of-flight mass spectrometry and nuclear magnetic resonance analysis. These analyses confirmed that the developed system was highly useful for production of D-BCAAs labeled with stable isotopes, making this the first reported enzymatic production of D-BCAAs labeled with stable isotopes. Our findings facilitate tracer studies investigating D-BCAAs and their derivatives.

Differences in sludge reduction effect due to chlorinated sites of dichlorophenol (DCP) isomers used in an uncoupling method were investigated. The study involved two different experiments. A batch experiment conducted to examine the effect of DCPs on SOUR clearly indicated that 3,5-DCP exhibited the strongest uncoupling activity. In the second experiment, in which six different sequence batch reactors were amended with 100 mM DCPs and operated for 2 months, 3,5-DCP also exhibited the strongest sludge reducing effect. The operations examined in this research maintained the concentration of DCPs at 100 mM, and reactors added with 3,5-DCP produced a COD removal rate of >80%, with almost no excess sludge generated in 2 months of continual operation. These results were not obtained with other DCPs. This is the first study to report differences in the sludge reduction effect associated with the chlorination site in the DCP isomer. The results indicate that 3,5-DCP is the most effective option for use in uncoupling methods involving DCPs.

Pseudomonas sp. strain CCA1 was isolated from leaf soil collected in Higashi-Hiroshima City in Hiroshima Prefecture, Japan. Here, we present a draft genome sequence of this strain. The genome consists of 24 contigs for a total of 6,993,992 bp, 8,917 predicted coding sequences, and a GC content of 67.2%.

Pyruvate plays an essential role in the central carbon metabolism of multiple organisms and is used as a raw material in the chemical, biochemical and pharmaceutical industries. To meet demand, large amounts of pyruvate are produced through fermentation processes. Here we describe a simple and efficient method for producing pyruvate in Escherichia coli. To stop carbon flux from pyruvate to fatty acids, the accBC genes, which encode the enzyme that catalyzes the first step of fatty acid biosynthesis and is essential for vegetative growth, were manipulated within the genome; its native promoter was replaced with the tetracycline (or doxycycline)-regulated promoter and the corresponding transcriptional regulator genes. The resulting strain grew normally in the presence of doxycycline, but showed poor growth upon withdrawal of doxycycline. Using this strain, we developed a high pyruvate producing strain (strain LAFCPCPt-accBC-aceE), in which the tetracycline-regulated promoter was also introduced upstream of aceE, and the ackA-pta, adhE, cra, ldhA, pflB and poxB genes were deleted. After determining the optimal culture conditions for this strain, the final pyruvate concentration reached 26.1 g L−1 after 72 h with a theoretical yield of 55.6 %. These levels are high enough to indicate that the developed strain has the potential for application to industrial production of pyruvate.

Burkholderia sp. strain CCA53 was isolated from leaf soil collected in Higashi-Hiroshima City in Hiroshima Prefecture, Japan. Here, we present a draft genome sequence of this strain, which consists of a total of 4 contigs containing 6,647,893 bp, with a G+C content of 67.0% and comprising 9,329 predicted coding sequences.

A thermostable, NADP(+)-dependent D: -amino acid dehydrogenase (DAADH) was created from the meso-diaminopimelate dehydrogenase of Ureibacillus thermosphaericus strain A1 by introducing five point mutations into amino acid residues located in the active site. The recombinant protein, expressed in Escherichia coli, was purified to homogeneity using a two-step separation procedure and then characterized. In the presence of NADP(+), the protein catalyzed the oxidative deamination of several D: -amino acids, including D: -cyclohexylalanine, D: -isoleucine and D: -2-aminooctanoate, but not meso-diaminopimelate, confirming the creation of a NADP(+)-dependent DAADH. For the reverse reaction, the corresponding 2-oxo acids were aminated in the presence of NADPH and ammonia. In addition, the D: -amino acid dehydrogenase showed no loss of activity at 65 °C, indicating the mutant enzyme was more thermostable than its parental meso-diaminopimelate dehydrogenase.

Microbial degradation of lignin releases fermentable sugars, effective utilization of which could support biofuel production from lignocellulosic biomass. In the present study, a lignin-degrading bacterium was isolated from leaf soil and identified as Burkholderia sp. based on 16S rRNA gene sequencing. This strain was named CCA53, and its lignin-degrading capability was assessed by observing its growth on medium containing alkali lignin or lignin-associated aromatic monomers as the sole carbon source. Alkali lignin and at least eight lignin-associated aromatic monomers supported growth of this strain, and the most effective utilization was observed for p-hydroxybenzene monomers. These findings indicate that Burkholderia sp. strain CCA53 has fragmentary activity for lignin degradation.