3-hydroxybutyrate Oligomers Research Articles

Optimization of Culture Conditions for Secretory Production of 3-Hydroxybutyrate Oligomers Using Recombinant Escherichia coli.

Poly(3-hydroxybutyrate) [P(3HB)] is the most representative polyhydroxyalkanoate (PHA), which is a storage polyester for prokaryotic cells. P(3HB)-producing recombinant Escherichia coli secretes diethylene glycol (DEG)-terminated 3HB oligomers (3HBO-DEG) through a PHA synthase-mediated chain transfer and alcoholysis reactions with externally added DEG. The purpose of this study was to optimize the culture conditions for the secretory production of 3HBO-DEG with jar fermenters. First, the effects of culture conditions, such as agitation speed, culture temperature, culture pH, and medium composition on 3HBO-DEG production, were investigated in a batch culture using 250-ml mini jar fermenters. Based on the best culture conditions, a fed-batch culture was conducted by feeding glucose to further increase the 3HBO-DEG titer. Consequently, the optimized culture conditions were reproduced using a 2-L jar fermenter. This study successfully demonstrates a high titer of 3HBO-DEG, up to 34.8 g/L, by optimizing the culture conditions, showing the feasibility of a new synthetic strategy for PHA-based materials by combining secretory oligomer production and subsequent chemical reaction.

Three new O-isocrotonyl-3-hydroxybutyric acid congeners produced by a sea anemone-derived marine bacterium of the genus Vibrio.

Liquid cultures of Vibrio sp. SI9, isolated from the outer tissue of the sea anemone Radianthus crispus, was found to produce three new O-isocrotonyl-3-hydroxybutyric acid derivatives, O-isocrotonyl-3-hydroxypentanoic acid (1), O-isocrotonyl-3-hydroxyhexanoic acid (2), and O-(Z)-2-hexenoyl-3-hydroxybutyric acid (3), together with the known O-isocrotonyl-3-hydroxybutyric acid (4). The structures of 1–3 were established by NMR spectroscopy and mass spectrometry, coupled with anisotropy-based chiral analysis, revealing the same R-configuration for all congeners 1–4. The compounds 1–4 were weakly growth-inhibitory against a marine fish ulcer pathogenic bacterium, Tenacibaculum maritimum NBRC16015. Structural similarities among 1–4, the O-isocrotonylated 3-hydroxybutyrate oligomers 5, and microbial biopolymer polyhydroxyalkanoates (PHA) suggest the presence of a common biosynthetic machinery, and hence a possible dehydrative modification at the hydroxy terminus of PHA.

Microbial Secretion Platform for 3-Hydroxybutyrate Oligomer and Its End-Capped Forms Using Chain Transfer Reaction-Mediated Polyhydroxyalkanoate Synthases.

The biodegradable polyester 3-hydroxybutyrate (3HB) polymer [P(3HB)] is intracellularly synthesized and accumulated in recombinant Escherichia coli. In this study, native polyhydroxyalkanoate (PHA) synthases are used to attempt to microbially secrete 3HB homo-oligomers (3HBOs), which are widely distributed in nature as physiologically active substances. High secretory production is observed, especially for the two PHA synthases from Aeromonas caviae and Bacillus cereus YB4. Surprisingly, an ethyl ester at the carboxy terminus (ethyl ester form) of 3HBOs is identified for most of the PHA synthases tested. Next, 3HBOs with a functional carboxyl group (carboxyl form of 3HBO) are obtained by using the alcohol dehydrogenase gene (adhE)-deficient mutant strain, suggesting that the endogenous ethanol produced in E. coli acts as a chain transfer (CT) agent in the generation of 3HBOs. Furthermore, an in vitro polymerization assay reveals that CT agents such as ethanol and free 3HB are involved in the generation of ethyl ester and carboxyl form of 3HBO, respectively. The microbial platform established herein allows the secretion of 3HBOs with desirable end structures by supplementation with various CT agents. The obtained 3HBOs and their end-capped forms may be used as physiologically active substances and building blocks for polymeric materials.

Deterioration of irradiation/high-temperature pretreated, linear low-density polyethylene (LLDPE) by Bacillus amyloliquefaciens

A Bacillus amyloliquefaciens strain was isolated from composted plastics and identified using microbial identification system BIOLOG and 16S rDNA sequences. The capability to attack virgin and ɣ-irradiation/high temperature-pretreated LLDPE films was investigated for comparison to evaluate the effect of pretreatment. A biodeteriorating effect characterized by low weight reductions of 1.1 ± 0.3 to 3.2 ± 1.3% was observed with the pretreated LLDPE within 40–60 days. The precision of the gravimetric method was sometimes negatively affected by the fragmentation of plastic films during the biological treatment or by traces of microbial biofilm firmly adhering to the plastic material. FTIR spectra before and after 60-day treatment indicated a decrease of carbonyl band and flattening of the 1300-1100 cm−1 zone due to bacterial action. GPC showed an increase of Mn and Mw of 2300–3700 and 32 200–35 500 g mol−1, respectively, and a decrease of polydispersity index suggesting presence of low molar weight LLDPE oligomers in pretreated LLDPE. The analysis of crystallinity and melting enthalpy detected the removal of oligomers during biodeterioration. ESI-MS analysis of the medium after 60-day biotreatment of pretreated LLDPE showed a presence of 3-hydroxybutyrate oligomers linked to the disappearance of low molar weight LLDPE oligomers that was not observed with virgin LLDPE. Coincidence of the removal of LLDPE oligomers and the appearance of 3-hydroxybutyrate oligomers suggested metabolization of low molecular LLDPE fractions present in the pretreated LLDPE by B. amyloliquefaciens. The experiments with virgin LLDPE demonstrated a positive effect of the pretreatment.

Methyl-esterified 3-hydroxybutyrate oligomers protect bacteria from hydroxyl radicals.

Bacteria rely mainly on enzymes, glutathione and other low-molecular weight thiols to overcome oxidative stress. However, hydroxyl radicals are the most cytotoxic reactive oxygen species, and no known enzymatic system exists for their detoxification. We now show that methyl-esterified dimers and trimers of 3-hydroxybutyrate (ME-3HB), produced by bacteria capable of polyhydroxybutyrate biosynthesis, have 3-fold greater hydroxyl radical-scavenging activity than glutathione and 11-fold higher activity than vitamin C or the monomer 3-hydroxybutyric acid. We found that ME-3HB oligomers protect hypersensitive yeast deletion mutants lacking oxidative stress-response genes from hydroxyl radical stress. Our results show that phaC and phaZ, encoding polymerase and depolymerase, respectively, are activated and polyhydroxybutyrate reserves are degraded for production of ME-3HB oligomers in bacteria infecting plant cells and exposed to hydroxyl radical stress. We found that ME-3HB oligomer production is widespread, especially in bacteria adapted to stressful environments. We discuss how ME-3HB oligomers could provide opportunities for numerous applications in human health.

Comparative proteome analysis reveals four novel polyhydroxybutyrate (PHB) granule-associated proteins in Ralstonia eutropha H16.

Identification of proteins that were present in a polyhydroxybutyrate (PHB) granule fraction isolated from Ralstonia eutropha but absent in the soluble, membrane, and membrane-associated fractions revealed the presence of only 12 polypeptides with PHB-specific locations plus 4 previously known PHB-associated proteins with multiple locations. None of the previously postulated PHB depolymerase isoenzymes (PhaZa2 to PhaZa5, PhaZd1, and PhaZd2) and none of the two known 3-hydroxybutyrate oligomer hydrolases (PhaZb and PhaZc) were significantly present in isolated PHB granules. Four polypeptides were found that had not yet been identified in PHB granules. Three of the novel proteins are putative α/β-hydrolases, and two of those (A0671 and B1632) have a PHB synthase/depolymerase signature. The third novel protein (A0225) is a patatin-like phospholipase, a type of enzyme that has not been described for PHB granules of any PHB-accumulating species. No function has been ascribed to the fourth protein (A2001), but its encoding gene forms an operon with phaB2 (acetoacetyl-coenzyme A [CoA] reductase) and phaC2 (PHB synthase), and this is in line with a putative function in PHB metabolism. The localization of the four new proteins at the PHB granule surface was confirmed in vivo by fluorescence microscopy of constructed fusion proteins with enhanced yellow fluorescent protein (eYFP). Deletion of A0671 and B1632 had a minor but detectable effect on the PHB mobilization ability in the stationary growth phase of nutrient broth (NB)-gluconate cells, confirming the functional involvement of both proteins in PHB metabolism.

Electrospray tandem mass spectrometry for the structural characterization of p-coumaric acid–oligo(3-hydroxybutyrate) conjugates

The synthesis of new biodegradable delivery system of p-coumaric acid (p-CA) for prospective application in cosmetology is described. p-Coumaric acid conjugates with oligo(3-hydroxybutyrate) (p-CA-OHB) were obtained via anionic ring opening oligomerization (ROO) of β-butyrolactone initiated by p-coumaric acid potassium salt. Electrospray ionization mass spectrometry (ESI-MS) analyses in positive-ion mode, supported by 1H NMR results, were used for the structural characterization at the molecular level of the obtained products. The obtained results indicated that the main product constitutes the conjugate of p-coumaric acid covalently bonded with oligo(3-hydroxybutyrate) chains through a hydrolysable ester bond. Moreover, application of ESI-MS allowed to detect two minor series of ions which corresponded to 3-hydroxybutyrate oligomers terminated by crotonate, hydroxyl and carboxyl end groups, respectively. Additionally, ESI-MS/MS tandem mass spectrometry studies validated the structure of the individual macromolecules of the obtained bioconjugate as well as the side products.

3-Hydroxybutyrate Oligomer Hydrolase and 3-Hydroxybutyrate Dehydrogenase Participate in Intracellular Polyhydroxybutyrate and Polyhydroxyvalerate Degradation in Paracoccus denitrificans

Genes encoding 3-hydroxybutyrate oligomer hydrolase (PhaZc) and 3-hydroxybutyrate dehydrogenase (Hbd) were isolated from Paracoccus denitrificans. PhaZc and Hbd were overproduced as His-tagged proteins in Escherichia coli and purified by affinity and gel filtration chromatography. Purified His-tagged proteins had molecular masses of 31 kDa and 120 kDa (a tetramer of 29-kDa subunits). The His-tagged PhaZc hydrolyzed not only 3-hydroxybutyrate oligomers but also 3-hydroxyvalerate oligomers. The His-tagged Hbd catalyzed the dehydrogenation of 3-hydroxyvalerate as well as 3-hydroxybutyrate. When both enzymes were included in the same enzymatic reaction system with 3-hydroxyvalerate dimer, sequential reactions occurred, suggesting that PhaZc and Hbd play an important role in the intracellular degradation of poly(3-hydroxyvalerate). When the phaZc gene was disrupted in P. denitrificans by insertional inactivation, the mutant strain lost PhaZc activity. When the phaZc-disrupted P. denitrificans was complemented with phaZc, PhaZc activity was restored. These results suggest that P. denitrificans carries a single phaZc gene. Disruption of the phaZc gene in P. denitrificans affected the degradation rate of PHA.

Properties of a Novel Intracellular Poly(3-Hydroxybutyrate) Depolymerase with High Specific Activity (PhaZd) in Wautersia eutropha H16

A novel intracellular poly(3-hydroxybutyrate) (PHB) depolymerase (PhaZd) of Wautersia eutropha (formerly Ralstonia eutropha) H16 which shows similarity with the catalytic domain of the extracellular PHB depolymerase in Ralstonia pickettii T1 was identified. The positions of the catalytic triad (Ser190-Asp266-His330) and oxyanion hole (His108) in the amino acid sequence of PhaZd deduced from the nucleotide sequence roughly accorded with those of the extracellular PHB depolymerase of R. pickettii T1, but a signal peptide, a linker domain, and a substrate binding domain were missing. The PhaZd gene was cloned and the gene product was purified from Escherichia coli. The specific activity of PhaZd toward artificial amorphous PHB granules was significantly greater than that of other known intracellular PHB depolymerase or 3-hydroxybutyrate (3HB) oligomer hydrolases of W. eutropha H16. The enzyme degraded artificial amorphous PHB granules and mainly released various 3-hydroxybutyrate oligomers. PhaZd distributed nearly equally between PHB inclusion bodies and the cytosolic fraction. The amount of PHB was greater in phaZd deletion mutant cells than the wild-type cells under various culture conditions. These results indicate that PhaZd is a novel intracellular PHB depolymerase which participates in the mobilization of PHB in W. eutropha H16 along with other PHB depolymerases.

Novel Intracellular 3-Hydroxybutyrate-Oligomer Hydrolase in Wautersia eutropha H16

Wautersia eutropha H16 (formerly Ralstonia eutropha) mobilizes intracellularly accumulated poly(3-hydroxybutyrate) (PHB) with intracellular poly(3-hydroxybutyrate) depolymerases. In this study, a novel intracellular 3-hydroxybutyrate-oligomer hydrolase (PhaZc) gene was cloned and overexpressed in Escherichia coli. Then PhaZc was purified and characterized. Immunoblot analysis with polyclonal antiserum against PhaZc revealed that most PhaZc is present in the cytosolic fraction and a small amount is present in the poly(3-hydroxybutyrate) inclusion bodies of W. eutropha. PhaZc degraded various 3-hydroxybutyrate oligomers at a high specific activity and artificial amorphous poly(3-hydroxybutyrate) at a lower specific activity. Native PHB granules and semicrystalline PHB were not degraded by PhaZc. A PhaZ deletion mutation enhanced the deposition of PHB in the logarithmic phase in nutrient-rich medium. PhaZc differs from the hydrolases of W. eutropha previously reported and is a novel type of intracellular 3-hydroxybutyrate-oligomer hydrolase, and it participates in the mobilization of PHB along with other hydrolases.

Properties of a Poly(3-hydroxybutyrate) Depolymerase from Penicillium funiculosum

A poly(3-hydroxybutyrate) (PHB) depolymerase was purified from a fungus, Penicillium funiculosum (IFO6345), with phenyl-Toyopearl and its properties were compared with those of other PHB depolymerases. The molecular mass of the purified enzyme was estimated at about 33 kDa by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The pH optimum and pI were 6.5 and 6.5, respectively. The purified protein showed affinity to Con A-Sepharose, indicating that it is a glycoprotein. Diisopropylfluorophosphate and dithiothreitol inhibited the depolymerase activity completely. The N-terminal amino acid sequence of the purified enzyme was TALPAFNVNPNSVSVSGLSSGGYMAAQL, which contained a “lipase box” sequence. This purified enzyme is one of the extracellular PHB depolymerase which belong to serine esterase. The purified enzyme showed relatively strong hydrolytic activity against 3-hydroxybutyrate oligomers compared with its PHB-degrading activity. PHB-binding experiments showed that P. funiculosum depolymerase has the weakest affinity for PHB of all the depolymerases examined.

The dimer and trimer of 3-hydroxybutyrate oligomer as a precursor of ketone bodies for nutritional care.

Ketone bodies have been considered as a means of providing energy because of their good penetration and rapid diffusion in peripheral tissues. However, because the currently available form of 3-hydroxybu-tyrate is the sodium salt, the sodium load is problematic. To avoid it, a mixture of dimer and trimer has been prepared as a precursor of D-3-hydroxybutyrate. The purpose of this study was to investigate whether and how the solution would be converted to monomers. The plasma concentration of 3-hydroxybutyrate monomer was measured in 10 rats during infusion of dimer and trimer. Stepwise dilutions of the solution were incubated with serum and liver homogenates from five rats, serum samples from five volunteers, and a liver sample from one patient with liver injury. The solution also was incubated with carboxylesterase and triacylglycerol lipase. The concentration of monomer in the medium was measured after incubation. The plasma concentration of 3-hydroxybutyrate monomer reached 572 +/- 11 micromol/L 15 minutes after beginning infusion of the mixture at a rate of 25 micromol x kg(-1) x min(-1) and 270 +/- 40 micromol/L at a rate of 12.5 micromol x kg(-1) min(-1). The solution was converted completely to monomers when incubated with rat serum or liver homogenate for 10 minutes. The mixture also was hydrolyzed by human liver homogenate but not by serum. The dimer and trimer of 3-hydroxybutyrate can be converted rapidly to monomer in rat and human tissues. 3-Hydroxybutyrate oligomers could be an energy substrate for injured patients.

Cloning and characterization of the polyhydroxybutyrate depolymerase gene of Pseudomonas stutzeri and analysis of the function of substrate-binding domains.

The extracellular polyhydroxybutyrate (PHB) depolymerase gene (phaZPst) of Pseudomonas stutzeri was cloned and sequenced. phaZPst was composed of 1,728 bp encoding a protein of 576 amino acids. Analyses of the N-terminal amino acid sequence and the matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrum of the purified enzyme showed that the mature enzyme consisted of 538 amino acids with a deduced molecular mass of 57,506 Da. Analysis of the deduced amino acid sequence of the protein revealed a domain structure containing a catalytic domain, putative linker region, and two putative substrate-binding domains (SBDI and SBDII). The putative linker region was similar to the repeating units of the cadherin-like domain of chitinase A from Vibrio harveyi and chitinase B from Clostridium paraputrificum. The binding characteristics of SBDs to poly([R]-3-hydroxybutyrate) [P(3HB)] and chitin granules were characterized by using fusion proteins of SBDs with glutathione S-transferase (GST). These GST fusion proteins with SBDII and SBDI showed binding activity toward P(3HB) granules but did not bind on chitin granules. It has been suggested that the SBDs of the depolymerase interact specifically with the surface of P(3HB). In addition, a kinetic analysis for the enzymatic hydrolysis of 3-hydroxybutyrate oligomers of various sizes has suggested that the catalytic domain of the enzyme recognizes at least two monomeric units as substrates.