Isolation, draft genome sequence, and identification of Paenibacillus glycanilyticus subsp. hiroshimensis CCS26.

To isolate the useful strain for fermentation to produce bioactive compounds, we screened oligotrophic bacteria, and then strain CCS26 was isolated from leaf soil collected in Japan. This strain was capable of growth on low-nutrient medium. To elucidate the taxonomy of strain CCS26, we determined the 16S rRNA gene and draft genome sequences, respectively. A phylogenetic tree based on 16S rRNA gene sequences showed that strain CCS26 clustered with Paenibacillus species. The draft genome sequence of strain CCS26 consisted of a total of 90 contigs containing 6,957,994 bp, with a GC content of 50.8% and comprising 6,343 predicted coding sequences. Based on analysis of the average nucleotide identity with the draft genome sequence, the strain was identified as P. glycanilyticus subsp. hiroshimensis CCS26.

Isolation and draft genome sequence of Paenibacillus sp. CCS19.

Here, we describe the isolation and draft genome sequence of Paenibacillus sp. CCS19. Paenibacillus sp. CCS19 was isolated from leaf soil collected in Japan and identified based on similarity of the 16S rRNA sequence with related Paenibacillus type strains. The draft genome sequence of Paenibacillus sp. CCS19 consisted of a total of 107 contigs containing 6,816,589 bp, with a GC content of 51.5% and comprising 5,935 predicted coding sequences.

Crystal structure of metagenomic ß-glycosidase MeBglD2 in complex with various saccharides.

Metagenomic MeBglD2 is a glycoside hydrolase family 1 (GH1) ß-glycosidase that has ß-glucosidase, ß-fucosidase, and ß-galactosidase activities, and is highly activated in the presence of monosaccharides and disaccharides. The ß-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.

Structural basis for the catalytic mechanism of the glycoside hydrolase family 3 isoprimeverose-producing oligoxyloglucan hydrolase from Aspergillus oryzae.

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: Glc4 Xyl3 ). Trp515 of IpeA plays a critical role in XXXG recognition at positive subsites. In addition, docking simulation of IpeA-XXXG suggested that two Tyr residues (Tyr268 and Tyr445) are involved in the catalytic reaction mechanism of IpeA. Tyr268 plays an important role in product turnover, whereas Tyr445 stabilizes the acid/base Glu524 residue, which serves as a proton donor. Our findings indicate that the substrate recognition machinery of IpeA is specifically adapted to xyloglucan oligosaccharides.

Draft Genome Sequence of Klebsiella pneumoniae subsp. pneumoniae CCI2, Isolated from Leaf Soil.

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%.

Draft Genome Sequence of Enterobacter sp. AS-1, a Potential Eurytrophic Recombination Host.

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.

Isolation, draft genome sequencing and identification of Enterobacter roggenkampii CCI9.

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.

Draft Genome Sequence of Enterobacter oligotrophicus CCA3, Isolated from Leaf Soil.

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%.

Paenibacillus glycanilyticus subsp. hiroshimensis subsp. nov., isolated from leaf soil collected in Japan.

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.

Performance of Burkholderia multivorans CCA53 for ethyl red degradation.

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.

Characterization of an NAD(P)+-dependent meso-diaminopimelate dehydrogenase from Thermosyntropha lipolytica.

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.

Identification and functional characterization of NAD(P)+ -dependent meso-diaminopimelate dehydrogenase from Numidum massiliense.

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.

Deinococcus kurensis sp. nov., isolated from pond water collected in Japan.

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.

Draft genome sequence of Deinococcus sp. KR-1, a potential strain for palladium-leaching.

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.

IoGAS1, a GPI-Anchored Protein Derived from Issatchenkia orientalis, Confers Tolerance of Saccharomyces cerevisiae to Multiple Acids.

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.

Enterobacter oligotrophica sp. nov., a novel oligotroph isolated from leaf soil.

A novel oligotrophic bacterium, designated strain CCA6, was isolated from leaf soil collected in Japan. Cells of the strain were found to be a Gram-negative, non-sporulating, motile, rod-shaped bacterium. Strain CCA6 grew at 10-45°C (optimum 20°C) and pH 4.5-10.0 (optimum pH 5.0). The strain was capable of growth in poor-nutrient (oligotrophic) medium, and growth was unaffected by high-nutrient medium. The major fatty acid and predominant quinone system were C16:0 and ubiquinone-8. Phylogenetic analysis based on 16S rRNA gene sequences indicated strain CCA6 presented as a member of the family Enterobacteriaceae. Multilocus sequence analysis (MLSA) based on fragments of the atpD, gyrB, infB, and rpoB gene sequences was performed to further identify strain CCA6. The MLSA showed clear branching of strain CCA6 with respect to Enterobacter type strains. The complete genome of strain CCA6 consisted of 4,476,585 bp with a G+C content of 54.3% and comprising 4,372 predicted coding sequences. The genome average nucleotide identity values between strain CCA6 and the closest related Enterobacter type strain were <88.02%. Based on its phenotypic, chemotaxonomic and phylogenetic features, strain CCA6 (=HUT 8142T =KCTC 62525T ) can be considered as a novel species within the genus Enterobacter with the proposed name Enterobacter oligotrophica.

Pseudomonas humi sp. nov., isolated from leaf soil.

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).

Artificial Thermostable D-Amino Acid Dehydrogenase: Creation and Application.

Many kinds of NAD(P)+-dependent L-amino acid dehydrogenases have been so far found and effectively used for synthesis of L-amino acids and their analogs, and for their sensing. By contrast, similar biotechnological use of D-amino acid dehydrogenase (D-AADH) has not been achieved because useful D-AADH has not been found from natural resources. Recently, using protein engineering methods, an NADP+-dependent D-AADH was created from meso-diaminopimelate dehydrogenase (meso-DAPDH). The artificially created D-AADH catalyzed the reversible NADP+-dependent oxidative deamination of D-amino acids to 2-oxo acids. The enzyme, especially thermostable one from thermophiles, was efficiently applicable to synthesis of D-branched-chain amino acids (D-BCAAs), with high yields and optical purity, and was useful for the practical synthesis of 13C- and/or 15N-labeled D-BCAAs. The enzyme also made it possible to assay D-isoleucine selectively in a mixture of isoleucine isomers. Analyses of the three-dimensional structures of meso-DAPDH and D-AADH, and designed mutations based on the information obtained made it possible to markedly enhance enzyme activity and to create D-AADH homologs with desired reactivity profiles. The methods described here may be an effective approach to artificial creation of biotechnologically useful enzymes.

Complete Genome Sequence of Ureibacillus thermosphaericus A1, a Thermophilic Bacillus Isolated from Compost.

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.

Production of acetoin from hydrothermally pretreated oil mesocarp fiber using metabolically engineered Escherichia coli in a bioreactor system.

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.