Advances and needs for endotoxin-free production strains.

The choice of an appropriate microbial host cell and suitable production conditions is crucial for the downstream processing of pharmaceutical- and food-grade products. Although Escherichia coli serves as a highly valuable leading platform for the production of value-added products, like most Gram-negative bacteria, this bacterium contains a potent immunostimulatory lipopolysaccharide (LPS), referred to as an endotoxin. In contrast, Gram-positive bacteria, notably Bacillus, lactic acid bacteria (LAB), Corynebacterium, and yeasts have been extensively used as generally recognized as safe (GRAS) endotoxin-free platforms for the production of a variety of products. This review summarizes the currently available knowledge on the utilization of these representative Gram-positive bacteria for the production of eco- and bio-friendly products, particularly natural polyesters, polyhydroxyalkanoates, bacteriocins, and membrane proteins. The successful case studies presented here serve to inspire the use of these microorganisms as a main-player or by-player depending on their individual properties for the industrial production of these desirable targets.

Characterization and identification of the proteins bound to two types of polyhydroxyalkanoate granules in Pseudomonas sp. 61-3.

Pseudomonas sp. 61-3 accumulates two types of polyhydroxyalkanoates (PHAs), poly(3-hydroxybutyrate) [P(3HB)], and poly(3HB-co-3-hydroxyalkanoates) [P(3HB-co-3HA)], and some proteins associated with their PHA granules have been identified. To date, PhaFPs (GA36) and PhaIPs (GA18) were identified from P(3HB-co-3HA) granules. In this study, the gene encoding GA24 associated with P(3HB) granule was identified as phbPPs. PhbPPs was composed of 192 amino acids with a calculated molecular mass of 20.4 kDa and was assumed to be a phasin. phbFPs gene and unknown ORF were also found on phb locus. PhbFPs was anticipated to be the transcriptional repressor of phbPPs gene. PhbPPs was bound to the P(3HB-co-3HA) granules with 3HB composition of more than 87 mol%, and PhaIPs and PhaFPs were bound to the P(3HB-co-3HA) granules with 3HA (C6-C12) composition of more than 13 mol% in the producing cells, suggesting that localization of these proteins is attributed to the monomer compositions of the copolymers.

Effects of lithium on growth, maturation, reproduction and gene expression in the nematode Caenorhabditis elegans.

Lithium (Li) has been widely used to treat bipolar disorder, and industrial use of Li has been increasing; thus, environmental pollution and ecological impacts of Li have become a concern. This study was conducted to clarify the potential biological effects of LiCl and Li(2)CO(3) on a nematode, Caenorhabditis elegans as a model system for evaluating soil contaminated with Li. Exposure of C. elegans to LiCl and Li(2)CO(3) decreased growth/maturation and reproduction. The lowest observed effect concentrations for growth, maturation and reproduction were 1250, 313 and 10 000 µm, respectively, for LiCl and 750, 750 and 3000 µm, respectively, for Li(2)CO(3). We also investigated the physiological function of LiCl and LiCO(3) in C. elegans using DNA microarray analysis as an eco-toxicogenomic approach. Among approximately 300 unique genes, including metabolic genes, the exposure to 78 µm LiCl downregulated the expression of 36 cytochrome P450, 16 ABC transporter, 10 glutathione S-transferase, 16 lipid metabolism and two vitellogenin genes. On the other hand, exposure to 375 µm Li(2)CO(3) downregulated the expression of 11 cytochrome P450, 13 ABC transporter, 13 lipid metabolism and one vitellogenin genes. No gene was upregulated by LiCl or Li(2)CO(3). These results suggest that LiCl and Li(2)CO(3) potentially affect the biological and physiological function in C. elegans associated with alteration of the gene expression such as metabolic genes. Our data also provide experimental support for the utility of toxicogenomics by integrating gene expression profiling into a toxicological study of an environmentally important organism such as C. elegans.

Aerosol soluble proteins in Asian dust in southwestern Japan.

Aerosol proteins, as core biological components of bioaerosols, are garnering increasing attention due to their environmental significance, including their roles in atmospheric processes and associated health risks. However, observational data on the proteins are very limited, leaving their distribution and variation in the atmosphere poorly understood. To investigate the long-distance transport of proteins with Asian dust in the Northern Hemisphere middle latitude westerlies to remote downwind areas, we quantified the soluble proteins in aerosol particles, referred to as aerosol soluble proteins (ASPs), collected in the coastal city of Kumamoto, Japan, during the spring of 2023, when three dust events occurred. The concentration of ASPs ranged from 0.22 to 1.68 µg m-3, with an average concentration of 0.73 ± 0.36 µg m-3 under dust conditions and 0.31 ± 0.05 µg m-3 under non-dust conditions. During the dust periods, the largest concentration of ASPs (1.68 µg m-3) coincided with the peak concentration of suspended particulate matter, and the concentration strongly correlated with the mass concentration of particles larger than 2.5 µm, indicating a close dependence of ASPs on dust particles. Primary estimations indicated a dry deposition flux of ASPs at approximately 1.10 ± 0.87 mg m-2 d-1 under the dust conditions. These results prove that Asian dust efficiently transports proteins, facilitating their dispersion in the atmosphere.

Number size distribution of bacterial aerosols in terrestrial and marine airflows at a coastal site of Japan.

Size-differentiated concentration of bacterial aerosols is essential for investigating their dissemination via the atmosphere. In this study, the number size distribution of bacterial aerosols was measured at a coastal site in southwestern Japan (32.324°N, 129.993°E) using a size-segregated eight-stage (>11, 7.0-11, 4.7-7.0, 3.3-4.7, 2.1-3.3, 1.1-2.1, 0.65-1.1, and 0.43-0.65µm) sampler. The results showed that the distribution differed according to the source areas: terrestrial air, oceanic air, or a combination of the two. The distribution in the long-distance transported terrestrial air from the Asian continent was monomodal, with a peak of 3.3-4.7 µm. The distribution in local land breeze air was bimodal, with the peaks at 0.43-1.1 and 3.3-4.7 µm. A similar bimodal distribution was encountered when the local island air and long-distance transported terrestrial air mixed. In contrast, the size distribution did not show clear peaks in the air from either nearby or remote marine areas. According to the air mass backward trajectories, the further the distance the air moved in the 72 h before arriving at the site, the lower the concentration of total bacterial aerosols. The estimation of dry deposition fluxes of bacterial cells showed that the deposition was dominated by cells larger than 1.1 µm with a relative contribution from 70.5 % to 93.7 %, except for the local land breeze cases, where the contributions in the size ranges larger and smaller than 1.1 µm were similar. These results show the distinctive number size distributions and removal processes of bacterial aerosols in different types of air. In addition, they indicate that size-dependent characteristics of airborne bacteria should be considered when studying their activities and roles in the atmospheric environment.

Identification and characterization of bacteriocin biosynthetic gene clusters found in multiple bacteriocins producing Lactiplantibacillus plantarum PUK6.

The multiple bacteriocins produced by Lactiplantibacillus plantarum PUK6 isolated from misozuke-tofu (tofu pickled in miso) were identified as plantaricins A, EF, and NC8. The pln locus (21,847 bp) containing the three plantaricin structural genes and another newly found putative bacteriocin structural genes (orf1 and orf2) were determined, and a biosynthesis mechanism was proposed. Reverse transcription-PCR analysis revealed that orf1 and orf2, the putative two-peptide bacteriocin structural genes, were expressed after 8 h (logarithmic growth phase) and 20 h (stationary growth phase) of cultivation of the PUK6 strain. Additionally, the growth inhibition profile obtained using the chemically synthesized mature peptides of Orf1 and Orf2 (referred to as mOrf1 and mOrf2) showed that the equimolar mixture of mOrf1 and mOrf2 exhibited bactericidal effect against the indicator strain Latilactobacillus sakei subsp. sakei JCM 1157T. Furthermore, fluorescence microscopic analysis revealed disruption of the cell membranes. These findings indicate that orf1 and orf2 are structural genes encoding class IIb bacteriocins consisting of two peptides. Therefore, the novel bacteriocin encoded by plnPUK6α (orf1) and plnPUK6ß (orf2) genes was named plantaricin PUK6. Since the PUK6 strain produces multiple bacteriocins, when used as a starter culture, it could contribute to improving the shelf life of fermented foods and preventing the appearance of bacteriocin-resistant bacteria.

Cell growth and P(3HB) accumulation from CO2 of a carbon monoxide-tolerant hydrogen-oxidizing bacterium, Ideonella sp. O-1.

Cell growth and accumulation of polyhydroxybutyric acid, P(3HB), from CO(2) in autotrophic condition of a newly isolated hydrogen-oxidizing bacterium, the strain O-1, was investigated. The bacterium, which was deposited in the Japan Collection of Microorganisms as JCM17105, autotrophically grows by assimilating H(2), O(2), and CO(2) as substrate. 16S rRNA gene sequence of the bacterium was the closest to Ideonella dechloratans (99%). Specific growth rate of the strain O-1 was faster than a hydrogen-oxidizing bacterium, Ralstonia eutropha, which is well-known P(3HB)-producing microorganism. The strain O-1 is tolerant to high O(2) concentration and it can grow above 30% (v/v) O(2), while the growth of R. eutropha and Alcaligenes latus was seriously inhibited. In culture medium containing 1 g/L (NH(4))(2)SO(4), cell concentration of the strain O-1 and P(3HB) increased to 6.75 and 5.26 g/L, respectively. The content of P(3HB) in the cells was 77.9% (w/w). The strain O-1 was very tolerant to carbon monoxide (CO) and it grew even at 70% (v/v) CO, while the growth of R. eutropha and A. latus were seriously inhibited at 5% (v/v) CO. From these results, it is expected that the strain O-1 will be useful in the manufacture of P(3HB) because the industrial exhaust gas containing CO(2), H(2), and CO can be directly used as the substrate in the fermentation process.

RANKL-induced DC-STAMP is essential for osteoclastogenesis.

Osteoclasts are bone-resorbing, multinucleated giant cells that are essential for bone remodeling and are formed through cell fusion of mononuclear precursor cells. Although receptor activator of nuclear factor-kappaB ligand (RANKL) has been demonstrated to be an important osteoclastogenic cytokine, the cell surface molecules involved in osteoclastogenesis are mostly unknown. Here, we report that the seven-transmembrane receptor-like molecule, dendritic cell-specific transmembrane protein (DC-STAMP) is involved in osteoclastogenesis. Expression of DC-STAMP is rapidly induced in osteoclast precursor cells by RANKL and other osteoclastogenic stimulations. Targeted inhibition of DC-STAMP by small interfering RNAs and specific antibody markedly suppressed the formation of multinucleated osteoclast-like cells. Overexpression of DC-STAMP enhanced osteoclastogenesis in the presence of RANKL. Furthermore, DC-STAMP directly induced the expression of the osteoclast marker tartrate-resistant acid phosphatase. These data demonstrate for the first time that DC-STAMP has an essential role in osteoclastogenesis.

Enhancement of lactate fraction in poly(lactate-co-3-hydroxybutyrate) synthesized by Escherichia coli harboring the D-lactate dehydrogenase gene from Lactobacillus acetotolerans HT.

For enhancing the lactate (LA) fraction of poly(lactate-co-3-hydroxybutyrate)s [P(LA-co-3HB)s], an exogenous D-lactate dehydrogenase gene (ldhD) was introduced into Escherichia coli. Recombinant strains of E. coli DH5α, LS5218, and XL1-Blue harboring the ldhD gene from Lactobacillus acetotolerans HT, together with polyhydroxyalkanoate (PHA)-biosynthetic genes containing a lactate-polymerizing enzyme (modified PHA synthase) gene, accumulated the P(LA-co-3HB) copolymer from glucose under microaerobic conditions (100 strokes/min). The LA fraction of copolymers synthesized in the strains of DH5α, LS5218, and XL1-Blue were 19.8, 15.7, and 28.5 mol%, respectively, which were higher than those of the strains without the ldhD gene (<6.7 mol% of LA units). Introduction of the exogenous ldhD gene into E. coli strains resulted in an enhanced LA fraction in P(LA-co-3HB)s.

Biosynthesis of novel lactate-based polymers containing medium-chain-length 3-hydroxyalkanoates by recombinant Escherichia coli strains from glucose.

Novel lactate (LA)-based polymers containing medium-chain-length 3-hydroxyalkanoates (MCL-3HA) were produced in fadR-deficient Escherichia coli strains from glucose as the sole carbon source. The genes encoding LA and 3-hydroxybutyrate (3HB) monomers supplying enzymes [propionyl-CoA transferase (PCT), d-lactate dehydrogenase (D-LDH), ß-ketothiolase (PhaA), and NADPH-dependent acetoacetyl-CoA reductase (PhaB)], MCL-3HA monomers supplying enzymes [(R)-3-hydroxyacyl-ACP thioesterase (PhaG) and (R)-3-hydroxyacyl (3HA)-CoA ligase] via fatty acid biosynthesis pathway, and modified polyhydroxyalkanoate (PHA) synthase [PhaC1(STQK)] of Pseudomonas sp. 61-3 were introduced into E. coli LS5218. This resulted in the synthesis of a novel LA-based copolymer, P(LA-co-3HB-co-3HA). 1H-nuclear magnetic resonance (NMR) analysis revealed the composition of P(LA-co-3HB-co-3HA) to be 19.7 mol% LA (C3), 74.9 mol% 3HB (C4), and 5.4 mol% MCL-3HA units of C8 and C10. Furthermore, the recombinant E. coli CAG18497 strain carrying these genes, excluding the phaAB genes, accumulated P(92.0% LA-co-3HA) with a novel monomer composition containing C3, C8, C10, and C12. 13C-NMR analysis showed the existence of LA-3HA sequence in the polymer. The solvent cast film of P(92.0% LA-co-3HA) exhibited transparency similar to poly(lactic acid).

Biosynthesis of Polyhydroxyalkanoate from Steamed Soybean Wastewater by a Recombinant Strain of Pseudomonas sp. 61-3.

Pseudomonas sp. 61-3 accumulates a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and a random copolymer, poly(3-hydroxybutyrate-co-3-hydroxyalkanoate) [P(3HB-co-3HA)], consisting of 3HA units of 4-12 carbon atoms. Pseudomonas sp. 61-3 possesses two types of PHA synthases, PHB synthase (PhbC) and PHA synthases (PhaC1 and PhaC2), encoded by the phb and pha loci, respectively. The P(94 mol% 3HB-co-6 mol% 3HA) copolymer synthesized by the recombinant strain of Pseudomonas sp. 61-3 (phbC::tet) harboring additional copies of phaC1 gene is known to have desirable physical properties and to be a flexible material with moderate toughness, similar to low-density polyethylene. In this study, we focused on the production of the P(3HB-co-3HA) copolymer using steamed soybean wastewater, a by-product in brewing miso, which is a traditional Japanese seasoning. The steamed soybean wastewater was spray-dried to produce a powder (SWP) and used as the sole nitrogen source for the synthesis of P(3HB-co-3HA) by the Pseudomonas sp. 61-3 recombinant strain. Hydrolyzed SWP (HSWP) was also used as a carbon and nitrogen source. P(3HB-co-3HA)s with relatively high 3HB fractions could be synthesized by a recombinant strain of Pseudomonas sp. 61-3 (phbC::tet) harboring additional copies of the phaC1 gene in the presence of 2% glucose and 10-20 g/L SWP as the sole nitrogen source, producing a PHA concentration of 1.0-1.4 g/L. When HSWP was added to a nitrogen- and carbon-free medium, the recombinant strain could synthesize PHA without glucose as a carbon source. The recombinant strain accumulated 32 wt% P(3HB-co-3HA) containing 80 mol% 3HB and 20 mol% medium-chain-length 3HA with a PHA concentration of 1.0 g/L when 50 g/L of HSWP was used. The PHA production yield was estimated as 20 mg-PHA/g-HSWP, which equates to approximately 1.0 g-PHA per liter of soybean wastewater.

Characterization of antimicrobial substance from Lactobacillus salivarius KL-D4 and its application as biopreservative for creamy filling.

Lactobacillus salivarius KL-D4 isolated from duck intestine produced bacteriocin which was stable at high temperature and a wide pH range of 3-10. Its cell free supernatant at pH 5.5 exhibited wide inhibitory spectrum against both G+ and G- bacteria. The highest bacteriocin production was obtained in MRS broth supplemented with 0.5 % (w/v) CaCO3 at 6 h by gentle shaking. PCR walking using specific primers at the conserved region of class-II bacteriocin resulted in 4 known genes of kld1, kld2, kld3 and kld4 with 100 % similarity to genes encoding for salivaricin α, ß, induction peptide and histidine protein kinase of Lb. salivarius GJ-24 which did not previously report for bacteriocin characterization, while showing 94, 93, 59 and 62 % to other salivaricin gene cluster, respectively. The high activities of 25,600 AU/ml indicated a strong induction peptide expressed by kld3 which has low similarity to previous inducer reported. Based on operon analysis, only kld1, kld3 and kld4 could be expressed and subsequently elucidated that only salivaricin α like bacteriocin was produced and secreted out of the cells. Using protein purification, only a single peptide band obtained showed that this strain produced one bacteriocin which could be salivaricin α namely salivaricin KLD showing about 4.3 kDa on SDS-PAGE. Partial purification by 20 % ammonium sulfate precipitation of the product was tested on the artificial contamination of creamy filling by Bacillus cereus, Enterococcus faecalis, Pseudomonas stutzeri, Staphylococcus sp. and Stenotrophomonas sp. resulting the growth inhibitory efficiency of 4.45-66.9, 11.5-100, 100, 0-28.1 and 5-100 % respectively. Therefore, salivaricin KLD can be a tentative biopreservative for food industry in the future.

Identification of genes differentially expressed in osteoclast-like cells.

Homeostasis of the skeletal system is maintained by a balance between bone formation and resorption. The receptor activator of NF-kappaB ligand (RANKL) induces the differentiation of bone-resorbing cells, osteoclasts. To identify genes regulated during osteoclast differentiation, we constructed a subtraction cDNA library using a mouse RAW264 macrophage cell line that differentiates into osteoclast-like multinucleated cells after treatment with RANKL. Northern blot analysis showed that RANKL treatment upregulated expression of 17 genes. Among these were the genes for five H(+)-ATPase subunits, two chemokines, and the osteoclast marker cathepsin K. In addition, a mouse homolog of human dendritic cell (DC)-specific transmembrane protein (DCSTAMP), whose function in osteoclastogenesis was recently revealed, was also included in the induced genes. Characterization of these inducible genes will provide an insight into the biology of osteoclasts and the mechanism of bone-related diseases.

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) by recombinant Escherichia coli from glucose.

The polyhydroxyalkanoate (PHA) copolymers consisting of short-chain-length (scl) and medium-chain-length (mcl) monomers have various properties ranging from stiff to flexible depending on the molar fraction of the monomer compositions. It has been reported that PhaG, which is first known as a (R)-3-hydroxyacyl-acyl carrier protein (ACP)-CoA transferase, actually functions as a 3-hydroxyacyl-ACP thioesterase, and the product of PP0763 gene from Pseudomonas putida KT2440 has a (R)-3-hydroxyacyl (3HA)-CoA ligase activity (Wang et al., Appl. Environ. Microbiol., 78, 519-527, 2012). In this study, we found a new (R)-3HA-CoA ligase (the product of PA3924 gene) from Pseudomonas aeruginosa PAO. The PA3924 gene was coexpressed with PHA synthase 1 gene (phaC1Ps) and phaGPs gene from Pseudomonas sp. 61-3, and ß-ketothiolase gene (phbARe) and acetoacetyl-CoA reductase gene (phbBRe) from Ralstonia eutropha in Escherichia coli LS5218 at 25°C. As a result, the copolymer containing 94.6 mol% 3-hydroxybutyrate (3HB) and 5.4 mol% mcl-3-hydroxyalkanoates (3HA) consisting of C8, C10, C12 and C14 was synthesized by recombinant E. coli LS5218 from glucose as the sole carbon source. The concentration of P(3HB-co-3HA) (scl-co-mcl-PHA) synthesized by the recombinant E. coli LS5218 harboring phaC1Ps, phaGPs, phbABRe and the PA3924 genes was approximately 7-fold higher than that of the recombinant LS5218 harboring phaC1Ps, phaGPs, phbABRe and the PP0763 genes. The number-average molecular weight of the P(3HB-co-5.4% 3HA) copolymer was 233 × 10(3), which was relatively high molecular weight. In addition, the physical and the mechanical properties of the copolymer were demonstrated to improve the brittleness of P(3HB) homopolymer.

Toxicity evaluation of glyphosate agrochemical components using Japanese medaka (Oryzias latipes) and DNA microarray gene expression analysis.

Using glyphosate agrochemical components, we investigated their acute toxicity to juvenile Japanese medaka (Oryzias latipes) as well as their toxic impact at gene expression level on the liver tissues of adult medaka using DNA microarray. In our acute toxicity test, juvenile medaka were exposed for 96 hr to each of the following glyphosate agrochemical components: 10~160 mg/l of glyphosate, 1.25~20 mg/l of fatty acid alkanolamide surfactant (DA), and 12~416 mg/l of a fully formulated glyphosate herbicide. As a result, LC(50) values of glyphosate, DA, and the glyphosate herbicide were > 160 mg/l, 8.5 mg/l, and 76.8 mg/l, respectively. On the other hand, adult male medaka fish were exposed to each of the glyphosate agrochemical components for 48 hr at the following concentrations: 16 mg/l of glyphosate, 0.5 mg/l of DA, and 16 mg/l-glyphosate/0.5 mg/l-DA mixture. Interestingly, DNA microarray analysis revealed that there were no significant gene expression changes in the medaka liver after exposure to glyphosate. Nevertheless, 78 and 138 genes were significantly induced by DA and the glyphosate/DA mixture, respectively. Furthermore, we identified five common genes that were affected by DA and glyphosate/DA mixture. These results suggested that glyphosate itself possessed very low toxicity as previously reported by some researchers at least to the small laboratory fish, and the major toxicity of the glyphosate agrochemical resided mainly in DA and perhaps in unintentionally generated byproduct(s) of glyphosate-DA mixture.

DNA microarray analysis of genomic responses of yeast Saccharomyces cerevisiae to nickel chloride.

In order to investigate nickel toxicity against the yeast Saccharomyces cerevisiae, genomic responses to nickel chloride were examined using yeast DNA microarrays. Microarray analysis revealed that exposure to 25 mM nickel chloride for 2 hr induced changes in gene expression in S. cerevisiae. Nickel chloride increased expression levels in 601 genes and decreased expression levels in 696 genes in S. cerevisiae.

Isolation and characterization of polyhydroxyalkanoates inclusions and their associated proteins in Pseudomonas sp. 61-3.

Two types of polyester inclusions of poly(3-hydroxybutyrate) [P(3HB)] and poly(3HB-co-3-hydroxyalkanoates) [P(3HB-co-3HA)] were isolated from crude extract of Pseudomonas sp. 61-3. Proteins associated with each inclusion were separated by SDS-PAGE. PHA synthase 1 (PhaC1(Ps)), PhaF(Ps), and PhaI(Ps) were identified from P(3HB-co-3HA) inclusions by N-terminal amino acid sequences analyses, as well as PHB synthase (PhbC(Ps)) and 24-kDa unknown protein were identified from P(3HB) inclusions. The structural genes of PhaF(Ps) and PhaI(Ps) were located downstream of the pha locus. The relative PHA/PHB synthase activities of each inclusion were measured for various 3-hydroxyacyl-coenzyme As of 4-12 carbon atoms. Direct atomic force microscopy observation of P(3HB) and P(3HB-co-3HA) inclusions demonstrated that the two types of inclusions had different morphologies.