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This review highlights the recent investigations of class IV polyhydroxyalkanoate (PHA) synthases, the newest classification of PHA synthases. Class IV synthases are prevalent in organisms of the Bacillus genus and are composed of a catalytic subunit PhaC (approximately 40 kDa), which has a PhaC box sequence ([GS]-X-C-X-[GA]-G) at the active site, and a second subunit PhaR (approximately 20 kDa). The representative PHA-producing Bacillus strains are Bacillus megaterium and Bacillus cereus; the nucleotide sequence of phaC and the genetic organization of the PHA biosynthesis gene locus are somewhat different between these two strains. It is generally considered that class IV synthases favor short-chain-length monomers such as 3-hydroxybutyrate (C4) and 3-hydroxyvalerate (C5) for polymerization, but can polymerize some unusual monomers as minor components. In Escherichia coli expressing PhaRC from B. cereus YB-4, the biosynthesized PHA undergoes synthase-catalyzed alcoholytic cleavage using endogenous and exogenous alcohols. This alcoholysis is thought to be shared among class IV synthases, and this reaction is useful not only for the regulation of PHA molecular weight but also for the modification of the PHA carboxy terminus. The novel properties of class IV synthases will open up the possibility for the design of new PHA materials.

Polyhydroxyalkanoate (PHA) synthase from the bacterium Bacillus cereus YB-4 (PhaRC YB4 ), a Class IV PHA synthase, shows both PHA polymerization and alcoholysis activities. In PhaRC YB4 -expressing Escherichia coli in the presence of ethanol, biosynthesized PHA is subjected to an ethyl-esterification at the carboxy terminus as a result of alcoholytic cleavage of the PHA chain by PhaRC YB4 . In this study, we investigated the range of alcohol species available for alcoholysis by PhaRC YB4 using E. coli JM109 as an assay host by monitoring the molecular weight of PHA during the second step of a two-step cultivation. PhaRC YB4 utilized various alcohols other than ethanol for alcoholysis. Through the use of bifunctional compounds for alcoholysis, the PHA carboxy terminus could be modified with thiol, alkynyl, hydroxy, and benzyl groups, resulting in the functionalization of the PHA carboxy terminus. Furthermore, one-step production of carboxy-terminal functionalized PHA was developed using E. coli XL1-Blue as a production host.

Bacillus cereus and Bacillus megaterium both accumulate polyhydroxyalkanoate (PHA) but their PHA biosynthetic gene (pha) clusters that code for proteins involved in PHA biosynthesis are different. Namely, a gene encoding MaoC-like protein exists in the B. cereus-type pha cluster but not in the B. megaterium-type pha cluster. MaoC-like protein has an R-specific enoyl-CoA hydratase (R-hydratase) activity and is referred to as PhaJ when involved in PHA metabolism. In this study, the pha cluster of B. cereus YB-4 was characterized in terms of PhaJ’s function. In an in vitro assay, PhaJ from B. cereus YB-4 (PhaJYB4) exhibited hydration activity toward crotonyl-CoA. In an in vivo assay using Escherichia coli as a host for PHA accumulation, the recombinant strain expressing PhaJYB4 and PHA synthase led to increased PHA accumulation, suggesting that PhaJYB4 functioned as a monomer supplier. The monomer composition of the accumulated PHA reflected the substrate specificity of PhaJYB4, which appeared to prefer short chain-length substrates. The pha cluster from B. cereus YB-4 functioned to accumulate PHA in E. coli; however, it did not function when the phaJYB4 gene was deleted. The B. cereus-type pha cluster represents a new example of a pha cluster that contains the gene encoding PhaJ.

Polyhydroxyalkanoates (PHAs) are a group of microbial intracellular biopolyesters that have wide potential applications for the plastics industry. Here, we report the results of molecular analyses of PHA synthases from Halomonas species, which are halophilic eubacteria known as promising industrial PHA producers, but the PHA synthase gene of Halomonas has not been cloned previously. In this study, the putative PHA synthase genes (phaC1 and phaC2) from two Halomonas strains (a new isolate, Halomonas sp. O-1, and the genome-sequenced strain, Halomonas elongata DSM2581) were cloned and characterized. We identified a gene related to the H. elongata DSM2581 PHA synthase gene (phaC1He) in the Halomonas sp. O-1 genome (phaC1HO1). PHA synthases usually contain a lipase box-like sequence Gly-X-Cys-X-Gly in their active sites. However, the equivalent sequence found in PhaC1HO1 and PhaC1He was determined to be Ser-X-Cys-X-Gly, with serine replacing the first glycine. The cloned phaC1HO1 and phaC1He genes were found to be functional when expressed in Escherichia coli JM109 and Ralstonia eutropha PHB-4. Site-directed mutagenesis studies showed that the cysteine in the Ser-X-Cys-X-Gly sequence was the catalytic center of Halomonas PhaC1 proteins and that replacement of serine with glycine slightly affected PHA biosynthesis and copolymer composition. In addition, a second potential PHA synthase gene (phaC2) was cloned and its polymerization activity was evaluated. The results herein provide an important molecular basis for PHA production by Halomonas species.

Polyhydroxyalkanoate (PHA)-producing Bacillus strains express class IV PHA synthase, which is composed of the subunits PhaR and PhaC. Recombinant Escherichia coli expressing PHA synthase from Bacillus cereus strain YB-4 (PhaRC YB-4 ) showed an unusual reduction of the molecular weight of PHA produced during the stationary phase of growth. Nuclear magnetic resonance analysis of the low-molecular-weight PHA revealed that its carboxy end structure was capped by ethanol, suggesting that the molecular weight reduction was the result of alcoholytic cleavage of PHA chains by PhaRC YB-4 induced by endogenous ethanol. This scission reaction was also induced by exogenous ethanol in both in vivo and in vitro assays. In addition, PhaRC YB-4 was observed to have alcoholysis activity for PHA chains synthesized by other synthases. The PHA synthase from Bacillus megaterium (PhaRC Bm ) from another subgroup of class IV synthases was also assayed and was shown to have weak alcoholysis activity for PHA chains. These results suggest that class IV synthases may commonly share alcoholysis activity as an inherent feature.

Developing Escherichia coli strains that are tolerant to acetate toxicity is important in light of an increased interest in the efficient utilization of lignocellulosic biomass feedstocks for the biosynthesis of value-added products. In this study, four strains known to produce polyhydroxyalkanoates (PHAs) from the typical hemicellulosic sugar xylose were tested for their tolerance to acetate. E. coli RSC10 was found to be tolerant of acetate, both in growth and fermentation studies. In the presence of acetate the strain showed a >2-fold increase in overall yields compared to using xylose alone as the feedstock. More importantly, the strain was found to be able to utilize acetate as a feedstock for biosynthesis of PHAs, with complete depletion of acetate (25 mM) at 9 h when acetate was the sole feedstock. Higher concentrations of acetate showed greater inhibition of fermentation than growth with a reduction of 90% in PHA yields at 100 mM. Additionally, the present work provides data to support the potential of acetate as a modulator for the control of composition of PHAs that incorporate lactate (LA) monomers into the copolymer from hemicellulose derived sugars.

Many microorganisms harbor genes necessary to synthesize biodegradable plastics known as polyhydroxyalkanoates (PHAs). We surveyed a genomic database and discovered a new cluster of class IV PHA synthase genes (phaRC). These genes are different in sequence and operon structure from any previously reported PHA synthase. The newly discovered PhaRC synthase was demonstrated to produce PHAs in recombinant Escherichia coli.

Cells of Lactobacilli co-aggregated with Escherichia coli K-12 cells to form co-aggregates under mixed-culture conditions at 37 °C for 24 h. Co-aggregation was inhibited by sodium dodecyl sulfate but not by protease. E. coli deletion mutants of fimbriae formation and lipopolysaccharide (LPS) formation did not co-aggregate with Lactobacilli. These results showed that fimbriae and LPS are necessary for co-aggregation between Lactobacilli and E. coli.

We describe the characterization of polyhydroxyalkanoate (PHA)-producing bacteria isolated from an ammunition-polluted soil in Kitakyushu City, Japan. Over 270 strains were evaluated for PHA accumulation based on a colony staining method using Nile red. Of these, nine strains were selected based on the intensity of Nile red fluorescence and the cells were quantitatively analyzed for PHA by gas chromatography. PHA accumulation was observed in five strains, all of which are inferred to be close to the Bacillus cereus group according to 16S rDNA sequence analysis. Interestingly, these strains produced a PHA copolymer, poly(3-hydroxybutyrae-co-3-hydroxyvalerate) [P(3HB-co-3HV)], with a 3HV fraction up to 2 mol% with glucose as a carbon source. Further characterization was performed on one isolate, B. cereus YB-4. Gel permeation chromatography analysis revealed that the number of average molecular weights of PHA accumulated in B. cereus YB-4 drastically changed from 722,000 to 85,000 over a 72-h cultivation period. Furthermore, the PHA synthase genes were cloned and the deduced amino acid sequences were determined. This study provides new insights into PHA biosynthesis by members of the B. cereus group.

This study was designed to analyze the diversity of aerobically growing bacteria in a landfill area, compared to those from a forest and a cultivated area at four different depths. The viable cell number of aerobes in the incineration ash layer (3.5(+/-0.4)x10(2)/g) was 10(3)- to 10(4)-fold lower than those in the other areas. On 16S ribosomal DNA similarity analysis of a total of 727 colonies, only the class Bacilli was detected in the incineration ash layer whose pH was extremely high (12.8), while five to seven classes were detected in the forest and the cultivated area. Of the genus Bacillus, B. licheniformis and the recently discovered Bacillus were predominant in the incineration ash layer. These analyses indicate that the incineration ash layer of a landfill might be a source that includes valuable or hitherto unreported Bacillus species.

Polyhydroxyalkanoates (PHAs) are eco-friendly plastics that are synthesized by bacteria as an intracellular storage material. Ethanol is a key compound in molecular weight regulation of PHA during biosynthesis. In this study, three strains of Escherichia coli expressing PHA synthase (PhaCRe, PhaCAc or PhaCHe) were cultured to compare the effect of ethanol level on PHA molecular weight. As a result, it was revealed that PhaCAc tends to produce low molecular weight PHA due to its high ethanol sensitivity whereas PhaCHe produces high molecular weight PHA even in the presence of ethanol.

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.

To investigate the possibility of applying staphylococcal catalase to eliminate H2O2 in lactic acid fermentation, an intracellular catalase from Staphylococcus warneri ISK-1 which exhibited high catalase activity was purified to homogeneity in a six-step purification procedure. The subunit molecular mass of the purified enzyme determined under denaturing conditions was 64,000. The absorption spectrum of the catalase showed a soret band at 406 nm, indicating that the enzyme is a heme protein. Km and Vmax of the catalase for H2O2 were 59 mM and 42,700 U/mg, respectively. The catalase showed a broad optimum pH (5.5–9.5) and stability to organic solvents. We cloned a catalase gene from S. warneri ISK-1 genome. Nucleotide sequence analysis of a 2.2-kb DNA fragment revealed an open reading frame of 1515 bp, called katA, encoding a protein of 504 amino acid residues. The predicted amino acid sequence showed a high homology with other typical catalases. No similarities to bacterial catalase-peroxidase type enzymes were found.

The biofilm degradation of Aggregatibacter actinomycetemcomitans is essential as a complete periodontal disease therapy, and here we show the effects of potential probiotic bacteria such as Lactobacillus spp. for the biofilm of several serotypes of A. actinomycetemcomitans strains. Eight of the 13 species showed the competent biofilm degradation of ≥ 90% reduction in biofilm values in A. actinomycetemcomitans Y4 (serotype b) as well as four of the seven species for the biofilm of A. actinomycetemcomitans OMZ 534 (serotype e). In contrast, the probiotic bacteria did not have a big impact for the degradation of A. actinomycetemcomitans SUNY 75 (serotype a) biofilm. The dispersed A. actinomycetemcomitans Y4 cells through the biofilm detachment were still viable and plausible factors for the biofilm degradation were not due to the lactic acid and low pH conditions. The three enzymes, protease, lipase, and amylase may be responsible for the biofilm degradation; in particular, lipase was the most effective enzyme for the biofilm degradation of A. actinomycetemcomitans Y4 along with the protease activity which should be also important for the other serotypes. Remarkable lipase enzyme activities were detected from some of the potential probiotics and a supporting result using a lipase inhibitor presented corroborating evidence that lipase activity is one of the contributing factors for biofilm degradation outside of the protease which is also another possible factor for the biofilm of the other serotype of A. actinomycetemcomitans strains. On the other hand, the biofilm of A. actinomycetemcomitans SUNY 75 (serotype a) was not powerfully degraded by the lipase enzyme because the lipase inhibitor was slightly functional for only two of potential probiotics.

Class IV polyhydroxyalkanoate (PHA) synthase from Bacillus cereus YB-4 (PhaRC(YB4)) or B. megaterium NBRC15308(T) (PhaRC(Bm)) was expressed in Ralstonia eutropha PHB(-)4 to compare the ability to produce PHA and the substrate specificity of PhaRCs. PhaRC(YB4) produced significant amounts of PHA and had broader substrate specificity than PhaRC(Bm).

Polyhydroxyalkanoate (PHA)-producing Bacillus strains possess class IV PHA synthases composed of two subunit types, namely, PhaR and PhaC. In the present study, PHA synthases from Bacillus megaterium NBRC15308(T) (PhaRC(Bm)), B. cereus YB-4 (PhaRC(YB4)), and hybrids (PhaR(Bm)C(YB4) and PhaR(YB4)C(Bm)) were expressed in Escherichia coli JM109 to characterize the molecular weight of the synthesized poly(3-hydroxybutyrate) [P(3HB)]. PhaRC(Bm) synthesized P(3HB) with a relatively high molecular weight (M(n) = 890 × 10(3)) during 72 h of cultivation, whereas PhaRC(YB4) synthesized low-molecular-weight P(3HB) (M(n) = 20 × 10(3)). The molecular weight of P(3HB) synthesized by PhaRC(YB4) decreased with increasing culture time and temperature. This time-dependent behavior was observed for hybrid synthase PhaR(Bm)C(YB4), but not for PhaR(YB4)C(Bm). These results suggest that the molecular weight change is caused by the PhaC(YB4) subunit. The homology between PhaCs from B. megaterium and B. cereus YB-4 is 71% (amino acid identity); however, PhaC(YB4) was found to have a previously unknown effect on the molecular weight of the P(3HB) synthesized in E. coli.

IgA is thought to be critical for intestinal immune system. Generally, the effect of food components on intestinal immune system is evaluated by animal experiments. However, the development of simple screening method using human cell culture makes them more cost effective and high-throughput. Thus, we investigated the culture condition of the IgA-producing human myeloma cell line KHM-1B that has unstable culture properties such as cluster formation, resulting in rapid cell death. Considering high-throughput screening, the culture condition was examined in 96-well culture plates and the following parameters were periodically measured: 1. Time to form clusters, 2. Viability, and 3. IgA production. As a result, we obtained a clone which could grow well and produce IgA antibodies stably. The doubling time of this clone was about 72 h, and continuously generated IgA antibodies for 2 months. Then, in order to examine the effect of food components on IgA production using this culture system, various components such as ascorbic acid, raffinose, lactoferrin, fructooligosaccharides, casein tryptic digests, strawberry crude extracts, and IL-6 as a positive control were added to the KHM-1B cell culture medium. The extracts from several kinds of strawberry as well as IL-6 were found to promote IgA production in KHM-1B cells. These results might be useful for screening of food derived IgA-promoting factors.