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At least 19 recordsLinked to original sources
PubMed1995

Metabolic pathway for biosynthesis of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) from 4-hydroxybutyrate by Alcaligenes eutrophus.

Various aerobic Gram-negative bacteria have been examined for their ability to use 4-hydroxybutyrate and 1,4-butanediol as carbon source for growth. Alcaligenes eutrophus strains H16, HF39, PHB-4 and Pseudomonas denitrificans 'Morris' were not able to grow with 1,4-butanediol or 4-hydroxybutyrate. From A. eutrophus HF39 spontaneous primary mutants (e.g. SK4040) were isolated which grew on 4-hydroxybutyrate with doubling times of approximately 3 h. Tn5::mob mutagenesis of mutant SK4040 led to the isolation of two phenotypically different classes of secondary mutants which were affected in the utilization of 4-hydroxybutyrate. Mutants exhibiting the phenotype 4-hydroxybutyrate-negative did not grow with 4-hydroxybutyrate, and mutants exhibiting the phenotype 4-hydroxybutyrate-leaky grew at a significantly lower rate with 4-hydroxybutyrate. Hybridization experiments led to the identification of a 10-kbp genomic EcoRI fragment of A. eutrophus SK4040, which was altered in mutants with the phenotype 4-hydroxybutyrate-negative, and of two 1-kbp and 4.5-kbp genomic EcoRI fragments, which were altered in mutants with the phenotype 4-hydroxybutyrate-leaky. This 10-kbp EcoRI fragment was cloned from A. eutrophus SK4040, and conjugative transfer of a pVDZ'2 hybrid plasmid to A. eutrophus H16 conferred the ability to grow with 4-hydroxybutyrate to the wild type. DNA-sequence analysis of this fragment, enzymic analysis of the wild type and of mutants of A. eutrophus as well as of recombinant strains of Escherichia coli led to the identification of a structural gene encoding for a 4-hydroxybutyrate dehydrogenase which was affected by transposon mutagenesis in five of six available 4-hydroxybutyrate-negative mutants. Enzymic studies also provided evidence for the presence of an active succinate-semialdehyde dehydrogenase in 4-hydroxybutyrate-grown cells. This indicated that degradation of 4-hydroxybutyrate occurs via succinate semialdehyde and succinate and that the latter is degraded by the citric acid cycle. NMR studies of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) accumulated from 4-hydroxy [1-13C]butyrate or 4-hydroxy[2-13C]butyrate as substrate gave no evidence for a direct conversion of 4-hydroxybutyrate into 3-hydroxybutyrate and therefore supported the results of enzymic analysis.

Alcaligenes↗
PubMed1999

Production of poly(3-hydroxybutyric acid-co-4-hydroxybutyric acid) and poly(4-hydroxybutyric acid) without subsequent degradation by Hydrogenophaga pseudoflava.

A Hydrogenophaga pseudoflava strain was able to synthesize poly(3-hydroxybutyric acid-co-4-hydroxybutyric acid) [P(3HB-co-4HB)] having a high level of 4-hydroxybutyric acid monomer unit (4HB) from gamma-butyrolactone. In a two-step process in which the first step involved production of cells containing a minimum amount of poly(3-hydroxybutyric acid) [P(3HB)] and the second step involved polyester accumulation from the lactone, approximately 5 to 10 mol% of the 3-hydroxybutyric acid (3HB) derived from the first-step culture was unavoidably reincorporated into the polymer in the second cultivation step. Reincorporation of the 3HB units produced from degradation of the first-step residual P(3HB) was confirmed by high-resolution 13C nuclear magnetic resonance spectroscopy. In order to synthesize 3HB-free poly(4-hydroxybutyric acid) [P(4HB)] homopolymer, a three-stage cultivation technique was developed by adding a nitrogen addition step, which completely removed the residual P(3HB). The resulting polymer was free of 3HB. However, when the strain was grown on gamma-butyrolactone as the sole carbon source in a synthesis medium, a copolyester of P(3HB-co-4HB) containing 45 mol% 3HB was produced. One-step cultivation on gamma-butyrolactone required a rather long induction time (3 to 4 days). On the basis of the results of an enzymatic study performed with crude extracts, we suggest that the inability of cells to produce 3HB in the multistep culture was due to a low level of 4-hydroxybutyric acid (4HBA) dehydrogenase activity, which resulted in a low level of acetyl coenzyme A. Thus, 3HB formation from gamma-butyrolactone is driven by a high level of 4HBA dehydrogenase activity induced by long exposure to gamma-butyrolactone, as is the case for a one-step culture. In addition, intracellular degradation kinetics studies showed that P(3HB) in cells was completely degraded within 30 h of cultivation after being transferred to a carbon-free mineral medium containing additional ammonium sulfate, while P(3HB-co-4HB) containing 5 mol% 3HB and 95 mol% 4HB was totally inert in interactions with the intracellular depolymerases. Intracellular inertness could be a useful factor for efficient synthesis of the P(4HB) homopolymer and of 4HB-rich P(3HB-co-4HB) by the strain used in this study.

3-Hydroxybutyric Acid↗
PubMed1990

Viscoelastic relaxations and thermal properties of bacterial poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate).

3-Hydroxybutyrate-3-hydroxyvalerate (3HB-3HV) as well as 3-hydroxybutyrate-4-hydroxybutyrate (3HB-4HB) copolyesters have been investigated by differential scanning calorimetry, thermogravimetric analysis and dynamic mechanical spectroscopy, over a wide range of compositions (0-95 mol% 3HV; 0-82 mol% 4HB). Both series of isolated copolyesters are partially crystalline at all compositions. Quenched samples show a glass transition that decreases linearly with increasing co-monomer molar fraction, more markedly when the co-monomer is 4HB. Above Tg, all copolyesters, rich in 3HB units, show a cold crystallization phenomenon followed by melting, while at the other end crystallization on heating is observed only in 3HB-3HV copolymers. The viscoelastic spectrum, strongly affected by thermal history, shows two relaxation regions: the glass transition, whose location depends on copolymer type and composition, and a secondary dispersion region at low temperatures (-130/-80 degrees C). The latter results from a water-related relaxation analogous to that of P(3HB) and, in 3HB-4HB copolymers, from another overlapping absorption peak centered at -130 degrees C, attributed to local motion of the methylene groups in the linear 4HB units.

Calorimetry, Differential Scanning↗
PubMed2000

Regulating the molar fraction of 4-hydroxybutyrate in poly(3-hydroxybutyrate-4-hydroxybutyrate) biosynthesis by Ralstonia eutropha using propionate as a stimulator.

The regulation of the molar fraction of 4-hydroxybutyrate (4-HB) in the poly(3-hydroxybutyrate-4-hydroxybutyrate) [P(3HB-4HB)] biosynthesis by Ralstonia eutropha (formerly Alcaligenes eutrophus) was attempted by the supplemental addition of propionate. The molar fraction of 4-HB in P(3HB-4HB) was increased significantly from 12.3 to 51.8 mol% by the addition of a small amount of propionate along with gamma-butyrolactone commonly used as a precursor for the biosynthesis of P(3HB-4HB). The mechanism of regulation by propionate was investigated by measuring the variation of enzyme activities related to the biosynthesis of P(3HB-4HB) and the level of intermediate metabolite acetyl-CoA. PHB synthase activity was induced significantly by propionate, and the acetyl-CoA concentration also increased significantly due to the additional supply of propionate. The overflowing acetyl-CoA seems to cause an inhibitory effect on the ketolysis reaction catalysing the lysis of 4-hydroxybutyryl-CoA to two molecules of acetyl-CoA; consequently, the 4-HB fraction available for polymerization increased. Accordingly, the molar fraction of 4-HB in P(3HB-4HB) biosynthesis seems to be regulated by both an increased 4-HB fraction and an activated PHB synthase due to the supplemental addition of propionate as a stimulator.

Journal Article↗
PubMed1993

gamma-Hydroxybutyric acid induced spike and wave discharges in rats: relation to high-affinity [3H]gamma-hydroxybutyric acid binding sites in the thalamus and cortex.

gamma-Hydroxybutyric acid is a naturally occurring compound which induces bilaterally synchronous spike and wave discharges in rats. The gamma-hydroxybutyric acid model of absence seizures simulates clinical absence seizures behaviorally as well as electrographically. The present study was undertaken in order to establish the role of the high-affinity gamma-hydroxybutyric acid binding sites in the generation of gamma-hydroxybutyric acid-induced spike and wave discharges. Spike and wave discharges induced by gamma-hydroxybutyric acid were recorded with the aid of bipolar depth electrodes implanted in discrete regions of thalamus, cortex and hippocampus. In the present study we found that ventroposterolateral, ventroposteromedial, medial and the reticular nuclei of the thalamus discharged synchronously with the cortical generation of spike and wave discharges. In the cortex, the superficial layers (I-IV) of frontoparietal cortex generated spike and wave discharges, whereas no spike and wave discharges were recorded from deeper layers (V-VI) of frontoparietal cortex. At the onset of spike and wave discharges induced by gamma-hydroxybutyric acid, a rapid but reversible upregulation of gamma-hydroxybutyric acid binding sites was observed. This increased [3H]gamma-hydroxybutyric acid binding was characterized by an increase in the number of gamma-hydroxybutyric acid sites with no significant change in their affinity for gamma-hydroxybutyric acid. Moreover, the change in [3H]gamma-hydroxybutyric acid binding was observed only in those thalamic structures and cortical layers which were found to be involved in the generation of spike and wave discharges induced by gamma-hydroxybutyric acid. The CA3 field or dorsal hippocampus possesses the highest density of [3H]gamma-hydroxybutyric acid binding sites of all brain regions. However, no significant change in [3H]gamma-hydroxybutyric acid binding was observed in this region nor was the CA3 field involved in the generation of spike and wave discharges during gamma-hydroxybutyric acid-induced absence-like seizures. These findings confirm that gamma-hydroxybutyric acid-induced absence-like seizures originate from thalamocortical pathways and that the onset of gamma-hydroxybutyric acid-induced spike and wave discharges is directly related to the regulation of gamma-hydroxybutyric acid binding sites in those regions which constitute the involved thalamocortical loop.

Animals↗
PubMed1995

A chemiluminescence-flow injection analysis of serum 3-hydroxybutyrate using a bioreactor consisting of 3-hydroxybutyrate dehydrogenase and NADH oxidase.

We describe a simple method for the highly sensitive chemiluminescence--flow injection analysis of 3-hydroxybutyrate in serum using a bioreactor column consisting of the two immobilized enzymes, 3-hydroxybutyrate dehydrogenase and NADH oxidase. The method was based on measuring the level of chemiluminescence formed by the reaction of a luminol-hexacyanoferrate mixture with hydrogen peroxide. The hydrogen peroxide was produced by the NADH oxidase reaction from NADH which was formed in the conversion of 3-hydroxybutyrate to acetoacetate by the 3-hydroxybutyrate dehydrogenase reaction. Among three immobilized enzyme columns, a coimmobilized, small 3-hydroxybutyrate dehydrogenase/NADH oxidase bioreactor alone (2 x 20 mm i.d.) readily hydrolyzed all of the injected 3-hydroxybutyrate into acetoacetate, although 3-hydroxybutyrate dehydrogenase catalyzed the reversible reaction. The present method generated linearity of the data up to 1.5 mM 3-hydroxybutyrate with satisfactory precision, reproducibility, and accurate reaction recoveries. The results from 3-hydroxybutyrate correlated satisfactorily with those obtained by other well-established methods. The coimmobilized 3-hydroxybutyrate dehydrogenase/NADH oxidase reactor unit showed good operational stability over a 5-week period, during which it was repeatedly used for 1500 analyses.

3-Hydroxybutyric Acid↗
PubMed1987

Glomerular filtration rate is increased in man by the infusion of both D,L-3-hydroxybutyric acid and sodium D,L-3-hydroxybutyrate.

A high glomerular filtration rate (GFR) is often found early in insulin-dependent diabetes mellitus (IDDM). It has been suggested that high circulating glucose, glucagon, and GH levels could play a role in this increase in GFR. On the other hand, patients with IDDM in poor metabolic control also have high circulating ketone body levels. This study was undertaken to determine whether exogenous D,L-3-hydroxybutyric acid at two infusion rates (40 and 30 mumol kg-1 min-1) for 180 min altered renal plasma flow (RPF), GFR, and the excretion rate of total protein, beta 2-microglobulin, and albumin in 11 normal (N) subjects and 11 IDDM patients in whom euglycemia was achieved and maintained using the insulin-glucose clamp technique. RPF and GFR were measured by a priming-continuous infusion of [125I]hippurate and [51Cr]EDTA, respectively. The 40 mumol kg-1 min-1 D,L-3-hydroxybutyric acid infusion increased RPF and GFR in both N and IDDM subjects. Mean RPF increased from 588 +/- 78 (+/- SD) to 706 +/- 129 mL min-1 1.73 m-2 in N and from 671 +/- 101 to 781 +/- 99 in IDDM. GFR increased from 121 +/- 11 to 151 +/- 15 ml min-1 1.73 m-2 in N and from 136 +/- 11 to 191 +/- 16 in IDDM. The filtration fraction also was significantly higher in IDDM than in N during the D,L-3-hydroxybutyric acid infusion. The 30 mumol kg-1 min-1 D,L-3-hydroxybutyric acid infusion increased RPF and GFR to a somewhat lesser extent in both groups. D,L-3-hydroxybutyric acid infusions increased the tubular reabsorption rate of ketone bodies and sodium. The increase in tubular sodium reabsorption rate was correlated significantly to that in the tubular ketone body reabsorption rate. A significant decrease in urinary pH was found during the D,L-3-hydroxybutyric acid infusion. D,L-3-Hydroxybutyrate sodium salt (30 mumol kg-1 min-1) also was infused in 5 of the 11 diabetic patients. A similar increase in GFR and RPF occurred. Both total protein and beta 2-microglobulin, but not albumin, excretion rates increased during D,L-3-hydroxybutyric acid (40 mumol kg-1 min-1) infusion in N and IDDM subjects. D,L-3-Hydroxybutyric acid infusion did not change plasma glucagon, GH, or renin activity.(ABSTRACT TRUNCATED AT 400 WORDS)

3-Hydroxybutyric Acid↗
PubMed2000

Characterization of the discriminative stimulus effects of gamma-hydroxybutyric acid as a means for unraveling the neurochemical basis of gamma-hydroxybutyric acid actions and its similarities to those of ethanol.

The present paper reviews the drug discrimination studies, both from the literature and from this laboratory, conducted to investigate the pharmacological profile of the discriminative stimulus effects of gamma-hydroxybutyric acid. Collectively, the results of these studies suggest that: (1) the discriminative stimulus effects of gamma-hydroxybutyric acid are composed of different cues, each one being the effect of gamma-hydroxybutyric acid on a specific receptor system; (2) the proportion of each component cue varies as the training dose of gamma-hydroxybutyric acid is increased; (3) the gamma-aminobutyric acid B-mediated cue is a major ingredient of the mixed stimulus of gamma-hydroxybutyric acid, but it is more prominent at high training doses than at low training doses of gamma-hydroxybutyric acid; and (4) positive modulation of the gamma-aminobutyric acid A receptor is a relevant part of the discriminative stimulus effects of low gamma-hydroxybutyric acid doses. Finally, data indicating symmetrical generalization between the discriminative stimulus effects of a specific range of doses of gamma-hydroxybutyric acid and those of ethanol are discussed in regard to their further support of the hypothesis that gamma-hydroxybutyric acid may exert its antialcohol effects through a substitution mechanism.

Animals↗
PubMed1990

Stable isotope dilution analysis of 4-hydroxybutyric acid: an accurate method for quantification in physiological fluids and the prenatal diagnosis of 4-hydroxybutyric aciduria.

A quantitative assay for 4-hydroxybutyric acid was developed using D6-4-hydroxybutyric acid as an internal standard. 4-Hydroxybutyric acid was isolated by liquid chromatography and the amount quantified by selected ion monitoring, ammonia chemical ionization gas chromatography/mass spectrometry of the trimethylsilyl derivatives. The concentrations of 4-hydroxybutyric in control physiological fluids were: 2.64 +/- 3.46 mmol mol-1 creatinine in urine, 1.09 +/- 2.87 mumol l-1 in plasma, 0.98 +/- 1.17 mumol l-1 in cerebrospinal fluid and 1.28 +/- 0.47 mumol l-1 in amniotic fluid. The concentration of 4-hydroxybutyric acid in the amniotic fluid from a pregnancy at risk for 4-hydroxybutyric aciduria was 2.30 mumol l-1, indicating an unaffected fetus. The stable isotope dilution assay of 4-hydroxybutyric acid in physiological fluid samples is a rapid, sensitive and accurate method for quantification, as well as a valuable technique for the prenatal diagnosis of 4-hydroxybutyric aciduria.

Acidosis↗
PubMed1983

Gas-chromatographic measurement of 3-hydroxybutyrate and lactate in plasma and 3-hydroxybutyrate in whole blood.

We describe two simple, rapid, and sensitive gas-chromatographic methods. One is for determining 3-hydroxybutyrate and lactate in plasma, the other for determining 3-hydroxybutyrate in perchloric acid extracts of blood. Samples are purified by adsorption onto graphitized carbon black (Carbopak B). After desorption, the two hydroxyacids are gas-chromatographically measured. Chromatography is on Carbopack B/polyethylene glycol (Mr 20 000)/trimesic acid, 94.4/ 4.8/0.8 by weight. A series of determinations of 3-hydroxybutyrate in whole blood from 15 subjects gave a mean of 59 (SD 46) mumol/L, with 97.8% analytical recovery (range 94.4-101.8%). In 15 plasma samples, we determined a mean value for 3-hydroxybutyrate of 62 (SD 72) mumol/L, with an analytical recovery of 96.4% (range 93.5-97.6%) and a mean value for lactate of 0.99 (SD 0.55) mmol/L, with an analytical recovery of 92.0 (range 91.3-97.6%). Six replicate analyses of plasma containing typical concentrations of lactate and 3-hydroxybutyrate gave respective CVs of 3.2, 2.1, 1.6, and 1.4%, and 4.9, 3.7, 2.8, and 2.0% for low, medium, high, and very high concentrations. Total analysis time for either method is less than 1 h.

3-Hydroxybutyric Acid↗
PubMed1994

Microbial synthesis and properties of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) in Comamonas acidovorans.

Comamonas acidovorans DS-17 was isolated from activated sludge and found to produce copolymers of 3-hydroxybutyrate (3HB) and 4-hydroxybutyrate (4HB) at 30 degrees C under growth-limited conditions. When 1,4-butanediol or 4-hydroxybutyric acid was used as the sole carbon source, a P(4HB) homopolymer was produced. Random copolymers of 3HB and 4HB units were produced on the addition of glucose or 3-hydroxybutyric acid to the culture solution of 4-hydroxybutyric acid. The physical properties of P(3HB-co-4HB) copolyesters with high 4HB fractions (64-100 mol%) were investigated. The copolyester films with high 4HB fractions exhibited the characteristics of a thermoplastic elastomer, and the tensile strength increased from 17 to 104 MPa as the 4HB fraction was increased from 64 to 100 mol%. The biodegradabilities of P(3HB-co-4HB) films were studied in aqueous solutions of extracellular polyhydroxybutyrate (PHB) depolymerase from Alcaligenes faecalis or of lipase from Rhizopus delemer. The erosion rate of P(3HB-co-4HB) films by PHB depolymerase decreased as the 4HB fraction in copolyester was increased from 64 to 100 mol%. In contrast, the erosion rate of films by lipase increased with the 4HB fraction.

Biodegradation, Environmental↗
PubMed1986

Degradation of poly(3-hydroxybutyrate) by poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis T1.

The extracellular poly(3-hydroxybutyrate) depolymerase purified from Alcaligenes faecalis T1 has two disulfide bonds, one of which appears to be necessary for the full enzyme activity. This depolymerase hydrolyzed not only hydrophobic poly(3-hydroxybutyrate) but also water-soluble trimer and larger oligomers of D-(-)-3-hydroxybutyrate, regardless of their solubilities in water. Kinetic analyses with oligomers of various sizes indicated that the substrate cleaving site of the enzyme consisted of four subsites with individual affinities for monomer units of the substrate. Analyses of the hydrolytic products of oligomers, which had labeled D-(-)-3-hydroxybutyrate at the hydroxy terminus, showed that the enzyme cleaved only the second ester linkage from the hydroxy terminus of the trimer and tetramer, and acted as an endo-type hydrolase toward the pentamer and higher oligomers. The enzyme appeared to have a hydrophobic site which interacted with poly(3-hydroxybutyrate) and determined the affinity of the enzyme toward the hydrophobic substrate.

Alcaligenes↗
PubMed1997

Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) in recombinant Escherichia coli grown on glucose.

A recombinant Escherichia coli strain has been developed that produces poly(3-hydroxybutyrate-co-4-hydroxybutyrate) when grown in complex medium containing glucose. This has been accomplished by introducing into E. coli DH5 alpha separate plasmids harboring the polyhydroxyalkanoate (PHA) biosynthesis genes from Ralstonia eutropha (formerly named Alcaligenes eutrophus) and the succinate degradation genes from Clostridium kluyveri, respectively. Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) levels reached 50% of the cell dry weight and contained up to 2.8 mol.% 4-hydroxybutyrate. The molecular weight of the polymer was 1.8 x 10(6).

Acyltransferases↗
PubMed2001

Degradation of poly (3-hydroxybutyrate) and its copolymer poly (3-hydroxybutyrate-co-3-hydroxyvalerate) by a marine Streptomyces sp. SNG9.

A marine Streptomyces sp. SNG9 was characterized by its ability to utilize poly(3-hydroxybutyrate) (PHB) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate P (3HB-co-HV). The bacterium grew efficiently in a simple mineral liquid medium enriched with 0.1 % poly(3-hydroxybutyrate) powder as the sole carbon source. Cells excreted PHB depolymerase and degraded the polymer particles to complete clarity in 4 days. The degradation activity was detectable by the formation of a clear zone around the colony (petri plates) or a clear depth under the colony (test tubes). The expression of PHB depolymerase was repressed by the presence of simple soluble carbon sources. Bacterial degradation of the naturally occurring sheets of poly(3-hydroxybutyrate) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was observed by scanning electron microscopy (SEM). Morphological alterations of the polymers sheets were evidence for bacterial hydrolysis.

Base Sequence↗
PubMed2000

Preparation of alkyl (R)-(-)-3-hydroxybutyrate by acidic alcoholysis of poly-(R)-(-)-3-hydroxybutyrate.

An efficient method for the preparation of optically active alkyl (R)-(-)-3-hydroxybutyrates by chemical depolymerization of biopolymer, poly-(R)-(-)-(3-hydroxybutyrate), was established. This method consists of simple recovery of poly-(R)-(-)-(3-hydroxybutyrate) from bacterial cells followed by acidic alcoholysis. When poly-(R)-(-)-(3-hydroxybutyrate) was purified by a simple digestion method that used 0.2 N sodium hydroxide, alkyl (R)-(-)-hydroxybutyrates were most efficiently produced by alcoholysis with anhydrous hydrochloric acid.

Journal Article↗
PubMed1999

Poly-3-hydroxybutyrate degradation in Rhizobium (Sinorhizobium) meliloti: isolation and characterization of a gene encoding 3-hydroxybutyrate dehydrogenase.

We have cloned and sequenced the 3-hydroxybutyrate dehydrogenase-encoding gene (bdhA) from Rhizobium (Sinorhizobium) meliloti. The gene has an open reading frame of 777 bp that encodes a polypeptide of 258 amino acid residues (molecular weight 27,177, pI 6.07). The R. meliloti Bdh protein exhibits features common to members of the short-chain alcohol dehydrogenase superfamily. bdhA is the first gene transcribed in an operon that also includes xdhA, encoding xanthine oxidase/dehydrogenase. Transcriptional start site analysis by primer extension identified two transcription starts. S1, a minor start site, was located 46 to 47 nucleotides upstream of the predicted ATG start codon, while S2, the major start site, was mapped 148 nucleotides from the start codon. Analysis of the sequence immediately upstream of either S1 or S2 failed to reveal the presence of any known consensus promoter sequences. Although a sigma54 consensus sequence was identified in the region between S1 and S2, a corresponding transcript was not detected, and a rpoN mutant of R. meliloti was able to utilize 3-hydroxybutyrate as a sole carbon source. The R. meliloti bdhA gene is able to confer upon Escherichia coli the ability to utilize 3-hydroxybutyrate as a sole carbon source. An R. meliloti bdhA mutant accumulates poly-3-hydroxybutyrate to the same extent as the wild type and shows no symbiotic defects. Studies with a strain carrying a lacZ transcriptional fusion to bdhA demonstrated that gene expression is growth phase associated.

Amino Acid Sequence↗
PubMed2000

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) formation from gamma-aminobutyrate and glutamate.

To provide 4-hydroxybutyryl-CoA for poly(3-hydroxybutyrate-co-4-hydroxybutyrate) formation from glutamate in Escherichia coli, an acetyl-CoA:4-hydroxybutyrate CoA transferase from Clostridium kluyveri, a 4-hydroxybutyrate dehydrogenase from Ralstonia eutropha, a gamma-aminobutyrate:2-ketoglutarate transaminase from Escherichia coli, and glutamate decarboxylases from Arabidopsis thaliana or E. coli were cloned and functionality tested by expression of single genes in E. coli to verify enzymatic activity, and uniquely assembled as operons under the control of the lac promoter. These operons were independently transformed into E. coli CT101 harboring the runaway replication vector pJM9238 for polyhydroxyalkanoate (PHA) production. Plasmid pJM9238 contains the PHA biosynthetic operon of R. eutropha under tac promoter control. Polyhydroxyalkanoate formation was monitored by nuclear magnetic resonance (NMR) spectroscopic analysis of the chloroform extracted and ethanol precipitated polyesters. Functionality of the biosynthetic pathway for copolymer production was demonstrated through feeding experiments using various carbon sources that supplied different precursors within the 4HB-CoA biosynthetic pathway.

Base Sequence↗
PubMed2006

Production and characterization of biodegradable terpolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate) by Alcaligenes sp. A-04.

The production of the terpolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate), P(3HB-co-3HV-co-4HB), by Alcaligenes sp. A-04 was investigated to determine the superior biodegradable polymer properties over those of poly(3-hydroxybutyrate), P(3HB), and its copolymers. The highest terpolymer content of 68% (w/w) was produced by Alcaligenes sp. A-04 at 60 h by shake-flask cultivation. The terpolymer with 93 mol% 4HB mole fraction units was produced when the cultivation time was extended to 96 h. Moreover, it was found that Alcaligenes sp. A-04 could utilize 1,4-butanediol for the synthesis of 3HB and 4HB monomers as well as the sodium salt of 4-hydroxybutyrate. The terpolymer content was 30% (w/w) and the composition was P(33%3HB-co-16%3HV-co-51%4HB). Next, terpolymers with 4HB mole fraction units ranging from 50 to 90 mol% were produced by varying the medium composition and cultivation time. The thermal and mechanical properties of the resulting terpolymers were different from those of the copolymers with a similar mole fraction of monomer units. The terpolymer P(4%3HB-co-3%3HV-co-93%4HB) showed an elongation of 430%, a toughness of 33 MPa, and Young's modulus of 127 MPa similar to those of low-density polyethylene. The terpolymer P(11%3HB-co-34%3HV-co-55%4HB) showed Young's Modulus of 618 MPa similar to that of polypropylene.

Alcaligenes↗