Expression and characterization of glutamate decarboxylase from <Emphasis Type="Italic">Lactobacillus brevis</Emphasis> HYE1 isolated from <Emphasis Type="Italic">kimchi</Emphasis>

A putative gene (gadlbhye1) encoding glutamate decarboxylase (GAD) was cloned from Lactobacillus brevis HYE1 isolated from kimchi, a traditional Korean fermented vegetable. The amino acid sequences of GADLbHYE1 showed 48% homology with the GadA family and 99% identity with the GadB family from L. brevis. The cloned GADLbHYE1 was functionally expressed in Escherichia coli using inducible expression vectors. The expressed recombinant GADLbHYE1 was successfully purified by Ni–NTA affinity chromatography, and had a molecular mass of 54 kDa with optimal hydrolysis activity at 55 °C and pH 4.0. Its thermal stability was determined to be higher than that of other GADs from L. brevis, based on its melting temperature (75.18 °C). Kinetic parameters including K_m and V_max values for GADLbHYE1 were 4.99 mmol/L and 0.224 mmol/L/min, respectively. In addition, the production of gamma-aminobutyric acid in E. coli BL21 harboring gadlbhye1/pET28a was increased by adding pyridoxine as a cheaper coenzyme.     

<Emphasis Type="Italic">Zunongwangia flava</Emphasis> sp. nov., belonging to the family <Emphasis Type="Italic">Flavobacteriaceae</Emphasis>, isolated from <Emphasis Type="Italic">Salicornia europaea</Emphasis>

A yellow pigmented bacterium designated strain MBLN094^T within the family Flavobacteriaceae was isolated from a halophyte Salicornia europaea on the coast of the Yellow Sea. This strain was a Gram-stain negative, aerobic, non-spore forming, rod-shaped bacterium. Phylogenetic analysis of the 16S rRNA gene sequence of strain MBLN094^T was found to be related to the genus Zunongwangia, exhibiting 16S rRNA gene sequence similarity values of 97.0, 96.8, 96.4, and 96.3% to Zunongwangia mangrovi P2E16^T, Z. profunda SM-A87^T, Z. atlantica 22II14-10F7^T, and Z. endophytica CPA58^T, respectively. Strain MBLN094^T grew at 20?37°C (optimum, 25?30°C), at pH 6.0?10.0 (optimum, 7.0?8.0), and with 0.5?15.0% (w/v) NaCl (optimum, 2.0?5.0%). Menaquinone MK-6 was the sole respiratory quinone. The polar lipids were phosphatidylethanolamine, two unidentified aminolipids, and four unidentified lipids. Major fatty acids were iso-C_17:0 3-OH, summed feature 3 (C_16:1ω6c and/or C16:1 ω7c), and iso-C_15:0. The genomic DNA G + C content was 37.4 mol%. Based on these polyphasic taxonomic data, strain MBLN094^T is considered to represent a novel species of the genus Zunongwangia, for which the name Zunongwangia flava sp. nov. is proposed. The type strain is MBLN094^T (= KCTC 62279^T = JCM 32262^T).     

<Emphasis Type="Italic">Gracilibacillus kimchii</Emphasis> sp. nov., a halophilic bacterium isolated from kimchi

A novel halophilic bacterium, strain K7^T, was isolated from kimchi, a traditional Korean fermented food. The strain is Gram-positive, motile, and produces terminal endospores. The isolate is facultative aerobic and grows at salinities of 0.0–25.0% (w/v) NaCl (optimum 10–15% NaCl), pH 5.5–8.5 (optimum pH 7.0–7.5), and 15–42°C (optimum 37°C). The predominant isoprenoid quinone in the strain is menaquinone-7 and the peptidoglycan of the strain is meso-diaminopimelic acid. The major fatty acids of the strain are anteisio-C_15:0, iso-C_15:0, and, C_16:0 (other components were < 10.0%), while the major polar lipids are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, and three unidentified lipids. A phylogenetic analysis of 16S rRNA gene sequence similarity showed that the isolated strain was a cluster of the genus Gracilibacillus. High levels of gene sequence similarity were observed between strain K7^T and Gracilibacillus orientalis XH-63^T (96.5%), and between the present strain and Gracilibacillus xinjiangensis (96.5%). The DNA G+C content of this strain is 37.7 mol%. Based on these findings, strain K7^T is proposed as a novel species: Gracilibacillus kimchii sp. nov. The type strain is K7^T (KACC 18669^T; JCM 31344^T).     

Draft Genome Sequence of Staphylococcus vitulinus F1028, a Strain Isolated from a Block of Fermented Soybean

Staphylococcus vitulinus is a coagulase-negative staphylococcus in the family Staphylococcaceae. This report describes the draft genome sequence of S. vitulinus F1028, which was isolated from a traditional Korean soybean food (meju). This 2.56-Mbp genome sequence is the first S. vitulinus genome of a strain isolated from a fermented soybean product.     

Genome Sequence of Staphylococcus lentus F1142, a Strain Isolated from Korean Soybean Paste

This report describes the draft genome sequence of Staphylococcus lentus F1142, which was isolated from a Korean fermented soybean paste (doenjang). The draft genome sequence contained 2.79 Mbp with a G+C content of 31.8%; this is the first S. lentus genome to be reported.     

Genetic localization and heterologous expression of validamycin biosynthetic gene cluster isolated from Streptomyces hygroscopicus var. limoneus KCCM 11405 (IFO 12704)

The validamycin biosynthetic gene cluster was isolated from Streptomyces hygroscopicus var. limoneus KTCC 1715 (IFO 12704) using a pair of degenerated PCR primers designed from the sequence of AcbC, 2-epi-5-epi-valiolone synthase in the acarbose biosynthesis. The nucleotide sequence analysis of the 37-kb DNA region revealed 22 complete ORFs including vldA, the acbC ortholog. Located around vldA, vldB to K were predicted to encode adenyltransferase, kinase, ketoreductase (or epimerase/dehydratase), glycosyltransferase, aminotransferase, dehydrogenase, phosphatase/phosphomutase, glycosyl hydrolase, transport protein, and glycosyltransferase, respectively. Apparently absent were any regulatory components within the sequenced region. The disruption of vldA abolished the validamycin biosynthesis and the plasmid-based complementation with vldABC restored production to the vldA-mutant; this substantiated that vldABC are essential to validamycin biosynthesis. This finding enabled us to discover the complete validamycin biosynthetic cluster. The cosmid clone of pJWS3001 harboring the 37-kb DNA region conferred validamycin-accumulation to Streptomyces lividans, indicating that the entire gene cluster of validamycin biosynthesis had been isolated. Additionally, Streptomyces albus, transformed with pJWS3001, produced a high level of alpha-glucosidase inhibitory activity in a R2YE liquid culture, which highlights the portability of the cluster within Streptomyces. The product of vldI was characterized as a glucoamylase (kcat, 32 s(-1); K(m), 5 mg/ml of starch) that does not play any apparent role in the validamycin biosynthesis. In order to characterize the upstream region, a vldW knockout was achieved via gene-replacement. A phenotypic study of the resulting mutant revealed that vldW is not essential for the host's ability to control Pellicularia filamentosa growth. The current information suggests that vldA to vldH is the genetic region essential to validamycin biosynthesis. This promises excellent opportunities to elucidate biosynthetic route(s) to the validamycin complex and to engineer the pathway for industrial application.     

Production of a platelet aggregation inhibitor, salmosin, by high cell density fermentation of recombinant Escherichia coli

Optimal conditions for a high cell density fermentation were investigated in a recombinant Escherichia coli producing salmosin, a platelet aggregation inhibitor. The optimized carbon and nitrogen sources were glycerol 10 g/l, yeast extract 30 g/l, and bacto-tryptone 10 g/l, yielding the dry cell weight (DCW) of 10.61 g/l in a 500 ml flask culture. The late-stage induction with 1% L-arabinose in a 5 l jar fermentor showed the highest DCW of 65.70 g/l after 27 h of the fed-batch fermentation. Around 2,200 mg/l of the protein was expressed as an inclusion body that was then refolded to obtain the active salmosin of 96 mg/l. We also confirmed the inhibitory activity against platelet aggregation of the active salmosin from the high cell density fermentation.     

Genome mining in Streptomyces. Elucidation of the role of Baeyer-Villiger monooxygenases and non-heme iron-dependent dehydrogenase/oxygenases in the final steps of the biosynthesis of pentalenolactone and neopentalenolactone

The pentalenolactone biosynthetic gene clusters have been cloned and sequenced from two known producers of the sesquiterpenoid antibiotic pentalenolactone, Streptomyces exfoliatus UC5319 and Streptomyces arenae TU?469. The recombinant enzymes PenE and PntE, from S. exfoliatus and S. arenae, respectively, catalyze the flavin-dependent Baeyer-Villiger oxidation of 1-deoxy-11-oxopentalenic acid (7) to pentalenolactone D (8). Recombinant PenD, PntD, and PtlD, the latter from Streptomyces avermitilis, each catalyze the Fe(2+)-α-ketoglutarate-dependent oxidation of pentalenolactone D (8) to pentalenolactone E (15) and pentalenolactone F (16). Incubation of PenD, PntD, or PtlD with the isomeric neopentalenolactone D (9) gave PL308 (12) and a compound tentatively identified as neopentalenolactone E (14). These results are corroborated by analysis of the ΔpenD and ΔpntD mutants of S. exfoliatus and S. arenae, respectively, both of which accumulate pentalenolactone D but are blocked in production of pentalenolactone as well as the precursors pentalenolactones E and F. Finally, complementation of the previously described S. avermitilis ΔptlE ΔptlD deletion mutant with either penE or pntE gave pentalenolactone D (8), while complemention of the ΔptlE ΔptlD double mutant with pntE plus pntD or penE plus pntD gave pentalenolactone F (16).