Determination of the safety of Halomonas sp. KM-1-derived D-ß-hydroxybutyric acid and its fermentation-derived impurities in mice and Japanese adults.

The use of halophilic bacteria in industrial chemical and food production has received great interest because of the unique properties of these bacteria; however, their safety remains under investigation. Halomonas sp. KM-1 intracellularly stores poly-D-ß-hydroxybutyric acid under aerobic conditions and successively secretes D-ß-hydroxybutyric acid (D-BHB) under microaerobic conditions. Therefore, we tested the safety of Halomonas sp. KM-1-derived D-BHB and the impurities generated during D-BHB manufacturing at a 100-fold increased concentration in acute tests using mice and daily intake of 16.0 g D-BHB in Japanese adults for 12 weeks. In the mice test, there were no abnormalities in the body weights or health of mice fed the purified D-BHB or its impurities. In the Japanese adult test, blood parameters and body condition showed no medically problematic fluctuations. These findings indicate that Halomonas sp. KM-1 is safe and can be used for commercial production of D-BHB and its derivatives.

Daily Intake of D-ß-Hydroxybutyric Acid (D-BHB) Reduces Body Fat in Japanese Adult Participants: A Randomized, Double-Blind, Placebo-Controlled Study.

Currently, there is considerable interest in ketone metabolism owing to the benefits for human health. Conventionally, strict dietary restrictions on carbohydrates are required to increase plasma ketone levels, while supplementation with D-ß-hydroxybutyric acid (D-BHB) can easily increase plasma ketone levels. We hypothesized that a daily intake of D-BHB could promote weight loss, especially through fat reduction. Herein, D-BHB (OKETOATM) was produced via a proprietary fermentation process from sugar. In this randomized, double-blind, placebo-controlled study, we assessed the safety and fat-reduction effects after 12 wk of daily ingestion of D-BHB (2.9 g) in 22 healthy Japanese adults and 22 control participants. Blood samples were collected pre- and post-treatment. Blood chemistry, anthropometric variables, and the body composition of the participants were investigated. Data analysis revealed that visceral fat at 12 wk significantly decreased by 9.0 cm2 (p=0.037), as evidenced by analysis of covariance. Blood parameters and body condition showed no significant differences between the two groups, and the participants reported no adverse effects or discomfort. Furthermore, data were analyzed by regrouping the participants. After removing one suspicious diabetes participant, all others showed significant decreases in visceral fat, body weight, BMI, and fat weight. Additionally, those aged under 50 y old had significantly decreased abdominal circumference and body fat percentage, in addition to visceral fat, body weight, BMI, and fat weight. Overall, our findings indicate that daily D-BHB intake may reduce body fat without dieting or exercise intervention. This study was registered with the UMIN Clinical Trials Registry as UMIN000045322.

Biocharacteristics and draft genome sequence of Halomonas hydrothermalis C22, a pyruvate-producing halophilic bacterium isolated from a commercial Spirulina culture in Vietnam.

Halophilic bacteria are receiving increasing attention for industrial chemical production processes due to their unique properties. Herein, an alkaliphilic and halophilic bacterium was isolated from a commercial Spirulina culture at Nghe An province in Vietnam and found to secrete pyruvate. Pyruvate is widely used as a starting material in the industrial biosynthesis of pharmaceuticals, and is employed for production of crop protection agents, polymers, cosmetics, and food additives. Phenotypic and chemotaxonomic characterization, and the 16S rRNA gene sequence homology with Halomonas hydrothermalis strain DSM 15,725 (99.2%) predicted that the strain belongs to the Halomonas genus, thus we named this strain as H. hydrothermalis strain C22. We investigated the biocharacteristics and capacity of strain C22 and determined the draft genome sequence comprising 3,934,166 bp with a G + C content of 60.2% encoding 3,668 proteins, 58 tRNAs, 9 rRNAs, and 1 tmRNA. Maximal pyruvate secretion reached 51.1 g/l after 84 h of cultivation. The results will facilitate future studies on the genetic and metabolic diversity of halophilic bacteria and expand our understanding of important bioprocesses in this microorganism.

Efficient production and secretion of oxaloacetate from Halomonas sp. KM-1 under aerobic conditions.

The alkaliphilic, halophilic bacterium Halomonas sp. KM-1 can utilize glucose for the intracellular storage of the bioplastic poly-(R)-3-hydroxybutyric acid (PHB) and extracellular secretion of pyruvate under aerobic conditions. In this study, we investigated the effects of sodium chloride concentration on PHB accumulation and pyruvate secretion in the KM-1 strain and, unexpectedly, observed that oxaloacetate, an important intermediate chemical in the TCA cycle, glycogenesis, and aspartic acid biosynthesis, was secreted. We then further analyzed oxaloacetate productivity after changing the sodium chloride additive concentration, additive time-shift, and culture temperature. In 42-h batch-cultivation experiments, we found that wild-type Halomonas sp. KM-1 secreted 39.0 g/L oxaloacetate at a rate of 0.93 g/(L h). The halophilic bacteria Halomonas has already gained attention for industrial chemical-production processes owing to its unique properties, such as contamination-free culture conditions and a tolerance for high substrate concentrations. Moreover, no commercial scale oxaloacetate production was previously reported to result from bacterial fermentation. Oxaloacetate is an important intermediate chemical in biosynthesis and is used as a health food based on its role in energy synthesis. Thus, these data provided important insights into the production of oxaloacetate and other derivative chemicals using this strain.

Efficient production and secretion of pyruvate from Halomonas sp. KM-1 under aerobic conditions.

The alkaliphilic, halophilic bacterium Halomonas sp. KM-1 can utilize both hexose and pentose sugars for the intracellular storage of bioplastic poly-(R)-3-hydroxybutyric acid (PHB) under aerobic conditions. In this study, we investigated the effects of the sodium nitrate concentration on PHB accumulation in the KM-1 strain. Unexpectedly, we observed the secretion of pyruvate, a central intermediate in carbon- and energy-metabolism processes in all organisms; therefore, pyruvate is widely used as a starting material in the industrial biosynthesis of pharmaceuticals and is employed for the production of crop-protection agents, polymers, cosmetics, and food additives. We then further analyzed pyruvate productivity following changes in culture temperature and the buffer concentration. In 48-h batch-cultivation experiments, we found that wild-type Halomonas sp. KM-1 secreted 63.3 g/L pyruvate at a rate of 1.32 g/(L·h), comparable to the results of former studies using mutant and recombinant microorganisms. Thus, these data provided important insights into the production of pyruvate using this novel strain.

Efficient secretion of (R)-3-hydroxybutyric acid from Halomonas sp. KM-1 by nitrate fed-batch cultivation with glucose under microaerobic conditions.

To establish a sustainable society, commodity chemicals need to be developed from biomass resources. Recently, (R)-3-hydroxybutyric acid ((R)-3-HB), a monomer of bioplastic poly-(R)-3-hydroxybutyric acid (PHB), has attracted attention for its possible use in the chemical industry. Halophilic bacteria have been considered for bioprocess applications due to certain characteristics such as the ability to grow in media containing high levels of the starting carbon source and the ability to be rarely contaminated. A halophilic bacterium Halomonas sp. KM-1 stores PHB intracellularly under aerobic conditions and secretes (R)-3-HB under microaerobic conditions. In this study, we optimized culture conditions to maximize (R)-3-HB secretion by KM-1 cells. By a simple nitrate fed-batch cultivation, Halomonas sp. KM-1 secreted 40.3g/L (R)-3-HB with a productivity of 0.48g L(-1)h(-1) with 20% (w/v) glucose. This level is one of the highest recorded productivity of (R)-3-HB to date.

Metabolomics-based component profiling of Halomonas sp. KM-1 during different growth phases in poly(3-hydroxybutyrate) production.

To investigate the relationship between the production of poly(3-hydroxybutyrate) (PHB) and metabolic changes during different growth phases, a non-sterile batch fermentation process involving an alkalophilic and halophilic bacterium, Halomonas sp. KM-1, was used. Intracellular metabolites were analyzed using gas chromatography-mass spectrometry to characterize the metabolic profile. Significant changes relating to PHB production were observed in the TCA cycle, lipid-synthesis and amino acid biosynthetic pathways were found to shift dramatically between the exponential growth and stationary phases. During the stationary phase, 17 metabolites were upregulated and a cell dry mass of 17.8 g/L that included 44.8% PHB was observed at 24h in 5% glucose-supplemented cultures, whereas 11 metabolites were upregulated and a cell dry mass of 38.4 g/L that included 73.7% PHB was observed at 36 h in 10% glucose-supplemented cultures. This study provides pattern analysis of metabolite regulation during PHB accumulation, indicating that multicomponent and phase-specific mechanisms are involved.

Efficient secretion of (R)-3-hydroxybutyric acid from Halomonas sp. KM-1 cultured with saccharified Japanese cedar under microaerobic conditions.

In the presence of d-glucose, consumption of pentoses such as d-xylose is somewhat repressed by most bacteria. However, in Halomonas sp. KM-1, simultaneous utilization of a pure hexose and pentose for growth and PHB production has been observed. Moreover, this strain has been shown to preferentially utilize d-xylose from a mixture of hexose and pentose. In addition, the KM-1 strain produced (R)-3-hydroxybutyric acid ((R)-3-HB) by using saccharified Japanese cedar (Cryptomeria japonica) wood. The concentration of intracellular PHB after aerobic cultivation for 24h was 8.4 g/L, and after shifting to microaerobic conditions and further cultivation for 18 h, the concentration of (R)-3-HB in the medium reached 8.0 g/L. These results show that the KM-1 strain can efficiently utilize saccharified Japanese cedar and secreted (R)-3-HB under microaerobic conditions.

Efficient secreted production of (R)-3-hydroxybutyric acid from living Halomonas sp. KM-1 under successive aerobic and microaerobic conditions.

Production of (R)-3-hydroxybutyric acid [(R)-3-HB] by strain Halomonas sp. KM-1 under successive aeration conditions was investigated. The first aerobic condition allowed both cell growth and intracellular storage of poly-(R)-3-hydroxybutyric acid (PHB). The second microaerobic condition, achieved by reducing the culture agitation rate, lead to the degradation of PHB to (R)-3-HB. The amount of PHB stored in KM-1 cells after 48-h cultivation under aerobic conditions was 16.4 g/l. In contrast, after a shift from aerobic to microaerobic conditions and a further 18-h cultivation, PHB content in KM-1 cells decreased to 0.9 g/l. Numerous intracellular PHB-containing granules were observed in cells under aerobic conditions by electron microscopy. After the shift to microaerobic conditions, the number and size of granules were significantly reduced, in agreement with the degradation of prestored PHB. On the other hand, under microaerobic conditions, the concentration of (R)-3-HB in the medium reached a maximum of 15.2 g/l, indicating the production and extracellular secretion of (R)-3-HB as a result of PHB digestion. Notably, cell lysis was not observed during the successive aeration conditions as assessed by elution of genomic DNA to the culture supernatant, cell morphology observed by electron microscopy and counts of colony formation. In this simple system utilizing a change of aeration during cultivation of strain Halomonas sp. KM-1, we obtained one of the highest levels of microbiological production of (R)-3-HB reported to date.

Draft genome sequence of Halomonas sp. strain KM-1, a moderately halophilic bacterium that produces the bioplastic poly(3-hydroxybutyrate).

We report the draft genome sequence of Halomonas sp. strain KM-1, which was isolated in Ikeda City, Osaka, Japan, and which produces the bioplastic poly(3-hydroxybutyrate). The total length of the assembled genome is 4,992,811 bp, and 4,220 coding sequences were predicted within the genome. Genes encoding proteins that are involved in the production and depolymerization of poly(3-hydroxybutyrate) were identified. The identification of these genes might be of use in the production of the bioplastic poly(3-hydroxybutyrate) and its monomer 3-hydroxybutyrate.

Taxonomic characterization and metabolic analysis of the Halomonas sp. KM-1, a highly bioplastic poly(3-hydroxybutyrate)-producing bacterium.

In a brief previous report, the gram-negative moderately halophilic bacterium, Halomonas sp. KM-1, that was isolated in our laboratory was shown to produce the bioplastic, poly(3-hydroxybutyrate) (PHB), using biodiesel waste glycerol (Kawata and Aiba, Biosci. Biotechnol. Biochem., 74, 175-177, 2010). Here, we further characterized this KM-1 strain and compared it to other Halomonas strains. Strain KM-1 was subjected to a polyphasic taxonomic study. Strain KM-1 was rod-shaped and formed colonies on a plate that were cream-beige in color, smooth, opaque, and circular with entire edges. KM-1 grew under environmental conditions of 0.1%-10% (w/v) NaCl, pH 6.5-10.5 and at temperatures between 10°C and 45°C. The G+C content of strain KM-1 was 63.9 mol%. Of the 16 Halomonas strains examined in this study, the strain KM-1 exhibited the highest production of PHB (63.6%, w/v) in SOT medium supplemented with 10% glycerol, 10.0 g/L sodium nitrate and 2.0 g/L dipotassium hydrogen phosphate. The intracellular structures within which PHB accumulated had the appearance of intracellular granules with a diameter of approximately 0.5 µm, as assessed by electron microscopy. The intra- and extra-cellular metabolites of strain KM-1 were analyzed by capillary electrophoresis mass spectrometry. In spite of the high amount of PHB stored intra-cellularly, as possible precursors for PHB only a small quantity of 3-hydroxybutyric acid and acetyl CoA, and no quantity of 3-hydroxybutyl CoA, acetoacetyl CoA and acetoacetate were detected either intra- or extra-cellularly, suggesting highly efficient conversion of these precursors to PHB.

Post-translational modifications of pro-opiomelanocrtin related hormones in medaka pituitary based on mass spectrometric analyses.

Direct tissue matrix-assisted laser desorption ionization with time-of-flight mass spectrometry analysis provides a selective detection of mass profile for the peptides contained into cell secretory granules. By this mass spectrometry with slice of pituitary, two novel molecular forms of pro-opimelanocrtin related hormone were found in the orange-red strain medaka (Oryzias latipes var.). The structures of [N,O-diacetyl Serine(1), O-acetyl Serine(3)]-α-melanocyte-stimulating hormone (MSH) and [hydroxyproline(15)]-ß-MSH, together with [phosphoserine(15)]-corticotropin-like intermediate lobe peptide, were determined for the first time using a collision-induced dissociation with electrospray ionization mass spectrometry. A combination of mass spectrometry analyses is thus a powerful tool to lead to the elucidation of the post-translational processing from the pre-prohormone.

Poly(3-hydroxybutyrate) production by isolated Halomonas sp. KM-1 using waste glycerol.

Biodiesel fuel is favored as a type of carbon neutral energy. To popularize its usage, by-product waste glycerol utilization is a critical problem. We tried to isolate waste glycerol utilizing bacteria, and obtained the alkalo- and halophile bacteria Halomonas sp. KM-1. This strain produced bioplastic poly(3-hydroxybutyrate) (PHB) in a simple medium and diluted waste glycerol as a sole carbon source.

Transformation of Spirulina platensis strain C1 (Arthrospira sp. PCC9438) with Tn5 transposase-transposon DNA-cation liposome complex.

Spirulina platensis is one of the most commercially important species of microalgae. Thus, it is an attractive candidate for genetic manipulation and the development of novel practical applications. However, this process is hampered by the absence of a stable gene transfer system, specifically the limited number of suitable vectors and transformation methods available for this organism. Artificial transposon systems developed by extracting the essential elements from natural transposons have been extensively studied, and recently a mutated transposase and transposon system was reported to improve transformation efficiency by electroporation. We applied a modified transformation strategy using a natural Tn5 transposon, transposase, and cation liposome complex by electroporation to improve the transformation efficiency for Spirulina platensis strain C1 (Arthrospira sp. PCC9438). Aggregation of cells became visible after 3 weeks during 2.0 microg/ml chloramphenicol selection, and growth continued for more than 12 months. Transfected chloramphenicol acetyltransferase (CAT) genes were detected in the genomic DNA by Southern hybridization. Transformed cells demonstrated CAT activity, but non-transformed cells did not.

Escherichia coli can be transformed by a liposome-mediated lipofection method.

Transformation of Escherichia coli is a basic technique for genetic engineering. We used a liposome-mediated lipofection method to transform electrocompetent E. coli cells which has little natural competence of foreign DNA without electroporation treatment, and got transformants with simple and quick treatment by a plasmid or a transposon and transposase complex.