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Culture conditions for the mass production of three green algae, Chlorella sp., Dunaliella salina DCCBC2 and Dunaliella sp., were optimized using a response surface methodology (RSM). A central composite design was applied to investigate the effects of initial pH, nitrogen and phosphate concentrations on the cultivation of microalgae. The optimal growth conditions estimated from the design are as follows: Chlorella sp. (initial pH 7.2, ammonium 17 mM, phosphate 1.2 mM), D. salina DCCBC2 (initial pH 8.0, nitrate 3.3 mM, phosphate 0.0375 mM) and Dunaliella sp. (initial pH 8.0, nitrate 3.7 mM, phosphate 0.17 mM). Culturing the microalgae with the optimized conditions confirmed that the maximum growth rates were attained for these parameters. The optimum CO(2) concentrations of Chlorella sp., D. salina DCCBC2 and Dunaliella sp. were 1.0, 3.0 and 1.0% (v/v), respectively. The specific growth rates (μ) of Chlorella sp., D. salina DCCBC2 and Dunaliella sp. were 0.58, 0.78 and 0.56 day(-1), respectively, and the biomass productivities were 0.28, 0.54 and 0.30 g dry cell wt l(-1) day(-1), respectively. The CO(2) fixation rates of Chlorella sp., D. salina DCCBC2 and Dunaliella sp. were 42.8, 90.9 and 45.5 mg l(-1) day(-1), respectively. Mixotrophic cultivation of Chlorella sp. with glucose increased biomass productivity from 0.28 to 0.51 g dry cell wt l(-1) day(-1). However, D. salina DCCBC2 and Dunaliella sp. were not stimulated by several organic compounds tested.

Methylophaga aminisulfidivorans MPT is a restricted facultatively marine methylotrophic bacterium that grows on methanol, methylated amines, dimethyl sulfide, and dimethyl sulfoxide. Here we present the highquality draft genome sequence of M. aminisulfidivorans MP T (KCTC 12909 T ‫؍‬ JCM 14647 T ), consisting of a chromosome (3,092,085 bp) and a plasmid (16,875 bp).The genus Methylophaga is a unique group of aerobic, halophilic, and non-methane-utilizing methylotrophs (6). Members of this genus have been isolated from various marine sediments and soda lakes and play an important role in the coastal environments participating in biogeochemical cycling of one-carbon substrates containing nitrogen, sulfur, or halogens (1, 7). They use the ribulose monophosphate pathway for one-carbon compound assimilation. The genus Methylophaga currently has seven species with validly published names, while only the genome sequence of Methylophaga thiooxydans (strain DMS010 T ) has been reported (3). Methylophaga aminisulfidivorans MPT (KCTC 12909 T ϭ JCM14647 T ) was isolated from seawater at Mokpo, South Korea (7). The strain is aerobic, neutrophilic, and halophilic and grows well on methanol, methylated amines, dimethyl sulfide, and dimethyl sulfoxide. Only fructose is utilized as a multicarbon source.The high-coverage genome sequence of M. aminisulfidivorans MP T was determined using a combination of Illumina Genome Analyzer IIx (150-bp single-end shotgun sequencing, 33,762,341 sequencing reads, 1,365-fold coverage) and the Roche Genome Sequencer FLX system (paired ends with an insert size of 3 kb, 909,213 reads, 60-fold coverage). Assembly of the Illumina sequencing reads was carried out with CLC genomics wb4 (CLCbio), whereas Newbler assembler 2.3 (Roche) was used to generate scaffolds from a 454-paired-end library. The two assemblies were merged into a single assembly using the CodonCode Aligner software (CodonCode Co.). After multiple rounds of gap-closing steps based on standard PCR and Sanger sequencing, 2 contigs (2,266,798 and 825,287 bp, respectively, GϩC content of 43.5 mol%) representing a chromosome and one circular plasmid (169,875 bp, GϩC content of 41.20 mol%) were obtained. The resultant genome sequence was uploaded into the RAST server (2) to predict the open reading frames (ORFs), tRNAs, and rRNAs. The predicted ORFs were annotated by searching against clusters of orthologous groups (8) and SEED databases (5).The chromosome of M. aminisulfidivorans MP T harbors 2,984 protein coding genes, 3 rRNA operons, and 31 tRNAs, whereas 190 coding genes and 9 tRNAs were found in the plasmid. The gene clusters responsible for methanol oxidation (mxaFJGIRSACKL) and synthesis of the cofactor pyrroloquinoline quinone (pqqBCDE) were identified from the draft genome sequence. Five types of cytochromes and one cytochrome c oxidase (cbb 3 ) were also predicted. A gene encoding a novel bacterial flavin-containing monooxygenase (MAMP_00532), which catalyzes nitrogen-containing compounds or indole by use of oxygen through an NADPH...

p-Hydroxybenzoate hydroxylase (pobA) and m-hydroxybenzoate hydroxylase (mobA) genes, from the moderate halophile Chromohalobacter sp. HS-2, were expressed and characterized. Solubilities of overexpressed recombinant MobA and PobA were enhanced by the induction of the heat-shock proteins DnaJ and DnaK. Each MobA and PobA maintained stable activity under high NaCl concentrations. V (max) and K (m) values for MobA with m-hydroxybenzoate were 70 μmol min(-1) mg(-1) protein and 81 μM, respectively. Similarly, those of PobA with p-hydroxybenzoate as substrate were 5 μmol min(-1) mg(-1) protein and 129 μM, respectively. The Escherichia coli expression system, including induction of heat shock proteins, was used to convert hydroxybenzoates into protocatechuate (3,4-dihydroxybenzoate) and revealed that resting cells harboring mobA converted 15 mM m-hydroxybenzoate to 15 mM protocatechuate while those harboring pobA converted 50 mM p-hydroxybenzoate to 35 mM protocatechuate at 30 °C, respectively.

Heterocapsa circularisquama RNA virus (HcRNAV) is the first single-stranded RNA virus to be characterized that infects dinoflagellates. The ability of HcRNAV coat protein (HcRNAV CP) to self-assemble into virus-like particles (VLPs) in vitro suggested that heterologous expression was possible, and that the VLPs might be ideal nanocontainers for the targeted delivery of genes and chemicals. In this paper, we report the expression of a codon-optimized HcRNAV 109 CP gene in Pichia pastoris and the production of self-assembled HcRNAV VLPs using large-scale fermentation. The HcRNAV 109 CP gene was synthesized according to the codon preference of P. pastoris and cloned into a pPICZA vector. The recombinant plasmid pPICZA-CPsyns was transformed into P. pastoris by electroporation. The resulting yeast colonies were screened by PCR and analyzed for protein expression by SDS polyacrylamide gel electrophoresis. After large-scale fermentation, the yield of HcRNAV CPsyns reached approximately 2.5 g L 1 within 4 d. The HcRNAV VLPs were purified using PEG precipitation followed by cesium chloride density gradient ultracentrifugation, and were subsequently analyzed using UV spectrophotometry and transmission electron microscopy. Fluorescence dye-labeled myoglobin was loaded into the cages of the HcRNAV VLPs and the encapsulation was confirmed by fluorescence spectroscopy. The results point to the possible utilization in pharmacology or nanotechnology of HcRNAV VLPs produced by P. pastoris fermentation. Heterocapsa circularisquama