Regulation of poly-β-hydroxybutyrate biosynthesis by nicotinamide nucleotide in Alcaligenes eutrophus

Abstract Poly-β-hydroxybutyrate biosynthesis was studied in Alcaligenes eutrophus under various nutrient-limiting conditions. When the cells were cultivated in nitrogen-limited media, both the levels of NAD(P)H and the ratios of NAD(P)H/NAD(P) were higher than those under nitrogen-sufficient conditions. The specific poly-β-hydroxybutyrate production rate was found to increase with the values of both NADH/NAD and NADPH/NADP, indicating that poly-β-hydroxybutyrate synthesis is directly regulated by the ratios of nicotinamide nucleotides. The effects of nicotinamide nucleotides on poly-β-hydroxybutyrate biosynthesis was investigated with regard to enzyme kinetics. Citrate synthase activity was significantly inhibited by NADH and NADPH, indicating that poly-β-hydroxybutyrate accumulation could be enhanced by facilitating the metabolic flux of acetyl-CoA to poly-β-hydroxybutyrate synthetic pathway. It was also found that cellular NADPH was a limiting substrate for NADPH-linked reductase, controlling the overall biosynthetic activity of poly-/3-hydroxybutyrate in this strain.     

Turnover of poly(3-hydroxybutyrate) (PHB) and its influence on the molecular mass of the polymer accumulated by Alcaligenes eutrophus during batch culture

Abstract Radiolabelled glucose was added to a batch culture of Alcaligenes eutrophus during the accumulation of poly(3-hydroxybutyrate) (PHB) to label newly synthesized polymer. The specific radioactivity of the polymer continued to increase, by approximately 30%, after the cessation of PHB accumulation, indicating that turnover of PHB was occurring. Fractionation of PHB showed that high molecular mass polymer was gradually replaced by PHB of lower molecular mass. Turnover of PHB is the cause of the slow decline in the molecular mass of PHB following the cessation of polymer accumulation but is unlikely to be the sole reason for the more rapid decrease in the molecular mass of PHB during the accumulation phase.     

Intracellular degradation of poly(3-hydroxybutyrate) granules of Zoogloea ramigera I-16-M

Abstract Intracellular degradation of poly(3-hydroxybutyrate) (PHB) in bacteria is not yet clear. The properties of the autodigestion of native PHB granules from Zooglea ramigera I-16-M were examined. The release of d (−)-3-hydroxybutyrate was observed only at pH values higher than about 8.5 and at relatively high ionic strength (optimal concentration 200 mM NaCl). Triton X-100 and diisopropylfluorophosphate inhibited this reaction. Addition of the supernatant fraction of Z. ramigera did not increase the release of d (−)-3-hydroxybutyrate from the native PHB granules. On the other hand, using the protease-treated PHB granules from Alcaligenes eutrophus as a substrate, PHB depolymerase activity was detected in the supernatant fraction of Z. ramigera cells. The soluble PHB depolymerase showed similar properties to the enzyme in the PHB granules. Since PHB depolymerase activity was found in fractions containing d (−)-3-hydroxybutyrate oligomer hydrolase activity, which were separated by DEAE-Toyopearl or by Sephacryl S-100, it is possible that the intracellular PHB depolymerase is identical to the oligomer hydrolase which has been purified already.     

Acid-tolerant poly(3-hydroxybutyrate) hydrolases from moulds

Abstract We have isolated some mould strains that can grow under acid conditions with poly(3-hydroxybutyrate) (PHB) as sole carbon source, and secrete PHB hydrolases active at pH values at least down to 3. An improved assay method for such enzymes using a pH stat has been developed, and used to determine the dependence of reaction rate on enzyme and polymer concentrations. The implications of these kinetic properties of the PHB hydrolase for its mode of action are discussed.     

Measurement of β-hydroxybutyrate in acute hyperketonemia in human brain

We report the measurement of D-beta-hydroxybutyrate (BHB) in the brains of six normal adult subjects during acute infusions of BHB. We used high field in vivo (1)H magnetic resonance (MR) spectroscopy in the occipital lobe in conjunction with an acute infusion protocol to elevate plasma BHB levels from overnight fasted levels (0.20 +/- 0.10 mM) to a steady state value of 2.12 +/- 0.30 mM. At this level of hyperketonemia, we determined a tissue BHB level of 0.24 +/- 0.04 mM. No increases in brain lactate levels were seen in these data. The concentrations of BHB and lactate were both considerably lower in comparison with previous data acquired in fasted adult subjects. This suggests that up-regulation of the monocarboxylic acid transporter occurs with fasting.     

Poly(β-hydroxyalkanoate) biosynthesis and accumulation by different Rhizobium species

Abstract Synthesis and accumulation of poly(β-hydroxy-alkanoate) in Rhizobium leguminosarum , R. leguminosarum biovar. trifolii, Rhizobium 'galega', Rhizobium 'hedysarum' and R. meliloti were studied.
Poly(β-hydroxybutyrate) is accumulated up to 55% of the cell dry weight. At 30–50% air saturation R. meliloti accumulates 50% of its biomass (5.5 g·1⁻¹ dry weight) as PHB after 90 h of batch fermentation. At 90% air saturation maximum accumulation (37.5%) of PHB occurs after 70 h cultivation.
R. meliloti strain 41 is able to synthesize the copolymer poly(β-hydroxybutyrate-co-β-hydroxyvalerate) at concentrations up to 58% of the biomass dry weight, containing up to 22 mol%β-hydroxyvalerate. Different concentrations of both copolymer and hydroxyvalerate were obtained when the microorganism was grown, in batch culture, in the presence of propionate or valerate and with glucose, sucrose or succinate as main carbon source.     

Differentiation of polyphosphate and poly-β-hydroxybutyrate granules in an Acinetobacter sp. isolated from activated sludge

Cells containing polyphosphate 71 micrograms P (mg protein)-1 and no poly-beta-hydroxybutyrate showed metachromatic granules but no lipid granules; cells containing poly-beta-hydroxybutyrate (15% of dry weight) showed fluorescence lipid granules but no metachromatic granules; whereas cells containing both polyphosphate and poly-beta-hydroxybutyrate showed both types of granules. These observations, together with a critical review of the literature, show a clear distinction between metachromatic (or volutin) granules and lipid granules.     

Isolation of poly(beta-L-malic acid)-degrading bacteria and purification and characterization of the PMA hydrolase from Comamonas acidovorans strain 7789

Several bacteria were isolated which were able to utilize poly(beta-L-malic acid) as sole carbon source for growth. The poly(beta-L-malic acid) hydrolyzing enzyme of Comamonas acidovorans strain 7789 was detected in the membrane fraction. The enzyme was purified by isolation of crude cell membranes by ultracentrifugation of disrupted cells, solubilization of the membrane fraction with octylglucoside, selective precipitation with 50% saturated ammonium sulfate and preparative isolectric focusing. SDS-PAGE analysis revealed a M(r) of 43,000. The pH optimum was 8.1 and the Km was 0.13 microM (in terms of monomeric units) and 0.0021 microM poly(beta-L-malic acid) at pH 8.1 (100 mM glycylglycine buffer). Addition of NaCl, KCl, CaCl2 or MgCl2 (from 25 to 100 mM) decreased the hydrolase activity, whereas EDTA or polymethane sulfonic acid fluoride had no influence on the enzyme. The depolymerization of poly(beta-L-malic acid) proceeded from the ends of the polyester resulting in the formation of L-malate. Esterase activity was not detectable with p-nitrophenyl acetate or p-nitrophenyl butyrate, which is used to determine for example poly(3-hydroxybutyric acid) depolymerase activity.     

Availability of neurotransmitter glutamate is diminished when β-hydroxybutyrate replaces glucose in cultured neurons

Ketone bodies serve as alternative energy substrates for the brain in cases of low glucose availability such as during starvation or in patients treated with a ketogenic diet. The ketone bodies are metabolized via a distinct pathway confined to the mitochondria. We have compared metabolism of [2,4-¹³C]β-hydroxybutyrate to that of [1,6-¹³C]glucose in cultured glutamatergic neurons and investigated the effect of neuronal activity focusing on the aspartate–glutamate homeostasis, an essential component of the excitatory activity in the brain. The amount of ¹³C incorporation and cellular content was lower for glutamate and higher for aspartate in the presence of [2,4-¹³C]β-hydroxybutyrate as opposed to [1,6-¹³C]glucose. Our results suggest that the change in aspartate–glutamate homeostasis is due to a decreased availability of NADH for cytosolic malate dehydrogenase and thus reduced malate–aspartate shuttle activity in neurons using β-hydroxybutyrate. In the presence of glucose, the glutamate content decreased significantly upon activation of neurotransmitter release, whereas in the presence of only β-hydroxybutyrate, no decrease in the glutamate content was observed. Thus, the fraction of the glutamate pool available for transmitter release was diminished when metabolizing β-hydroxybutyrate, which is in line with the hypothesis of formation of transmitter glutamate via an obligatory involvement of the malate–aspartate shuttle.     

Production of poly(3-hydroxybutyrate) from inexpensive substrates

Two inexpensive substrates, starch and whey were used to produce poly(3-hydroxybutyrate) (PHB) in fed-batch cultures of Azotobacter chroococcum and recombinant Escherichia coli, respectively. Oxygen limitation increased PHB contents in both fermentations. In fed-batch culture of A. chroococcum, cell concentration of 54 g l⁻¹ with 46% PHB was obtained with oxygen limitation, whereas 71 g l⁻¹ of cell with 20% PHB was obtained without oxygen limitation. The timing of PHB biosynthesis in recombinant E. coli was controlled using the agitation speed of a stirred tank fermentor. A PHB content of 80% could be obtained with oxygen limitation by increasing the agitation speed up to only 500 rpm.     

Structure of native poly(3-hydroxybutyrate) granules characterized by X-ray diffraction

The structure of native poly(3-hydroxybutyrate) (PHB) granules of Alcaligenes eutrophus was characterized in wet cells or wet granules by analysis of X-ray diffraction. The PHB granules in intact cells were completely amorphous, but became crystalline after treatment with alkali or sodium hypochlorite. The native PHB granules were isolated from the cells by treatment with enzymes and sonic oscillation. The isolated PHB granules remained amorphous in suspension. The PHB granules were crystallized by various treatments with aqueous acetone, alkaline solution (of either NaOH or sodium hypochlorite), and lipase in an aqueous environment. These results suggest that crystallization of PHB molecules is started by the removal of a lipid component from native granules by various treatments.     

Physiology and molecular genetics of poly(β-hydroxyalkanoic acid) synthesis in Alcaligenes eutrophus

The Alcaligenes eutrophus genes for beta-ketothiolase, NADPH-dependent acetoacetyl-CoA reductase and poly(beta-hydroxybutyric acid) synthase (PHB synthase) which comprise the three-step PHB-biosynthetic pathway, were cloned. Molecular studies revealed that these genes are organized in a single operon. The A. eutrophus PHB-biosynthetic genes are readily expressed in other bacteria, and DNA fragments harbouring the operon can be used as a cartridge to confer to other bacteria the ability to synthesize PHB from acetyl-CoA. The biochemical and physiological capabilities of A. eutrophus for the synthesis of a wide variety of polyhydroxyalkanoates are discussed.     

Effect of poly(3-hydroxybutyrate) (PHB) content on the starvation-survival of bacteria in natural waters

Abstract The effect of poly(3-hydroxybutyrate) (PHB) content on the survival of wild-type strains and PHB negative mutants of Bacillus megaterium and Alcaligenes eutrophus in natural waters was studied. The survival strategy of B. megaterium was dominated by the development of resistant forms, but the number of the wild-type vegetative cells was higher than that of PHB mutant strain. In some environmental conditions the mutant spores needed a heat shock for germination, a fact that suggests, for the first time, that PHB plays a role in this phenomenon. Survival of A. eutrophus wild-type strain in all experiments was higher compared to the PHB mutant, and differences were significant. In raw river water, survival of both species was lower than in sterile river water.     

β(1,3)-Glucanases from Geotrichum lactis: activity on its own nascent and preformed β(1,3)-glucan

Abstract Actively growing mycelium of Geotrichum lactis contains at least three β(1,3)-glucanase activities. Two of the activities have been characterized as exo- and the third as endo-hydrolytic. The action of the activities on β(1,3)-glucan synthesized in vitro by the β(1,3)-glucan synthase system from G. lactis has been studied. One of the exo-β-glucanases and the endo-β-glucanase were active on this β(1,3)-glucan and the degradation rates were higher on nascent than on preformed β(1,3)-glucan.     

Determination of the active sites serine of the poly (3-hydroxybutyrate) depolymerases of Pseudomonas lemoignei (PhaZ5) and of Alcaligenes faecalis

Abstract Mutational analysis of the poly(3-hydroxybutyrate) (PHB) depolymerase A of Pseudomonas lemoignei and of the poly(3-hydroxybutyrate) depolymerase of Alcaligenes faecalis revealed that S¹³⁸ ( P. lemoignei ) and S¹³⁹ ( A. faecalis ) are essential for activity. Both serines are part of a strictly conserved pentapeptide sequence which is present in all poly(3-hydroxybutyrate) depolymerases analyzed so far (G-L-S-S(A)-G) and which resembles the lipase box of lipases and other serine hydrolases (G-X-S-X-G). Mutation of another conserved serine, namely S¹⁹⁵ ( P. lemoignei ) and S¹⁹⁶ ( A. faecalis ), resulted in mutant proteinswith almost full activity and proved that S195 and S196 are not essential for activity. The results indicate the structural and functional relationship of poly(3-hydroxybutyrate) depolymerases to the family of serine hydrolases.     

Plant-Derived Neurotoxic Amino Acids (β-N-Oxalylamino-l-Alanine and β-N-Methylamino-l-Alanine): Effects on Central Monoamine Neurons

In the present study the subacute effects of beta-N-oxalylamino-L-alanine (BOAA) and beta-N-methylamino-L-alanine (BMAA) on CNS monoamine neurons in rats were investigated following intracisternal injections or local intracerebral administration into substantia nigra. In vitro effects of BOAA and BMAA on high-affinity synaptosomal uptake of dopamine (DA), noradrenaline (NA), and serotonin (5-HT) were also examined. Intracisternal administration of BMAA decreased NA levels in hypothalamus, whereas no effects were seen on DA or 5-HT levels. Following intranigral injections of BOAA, NA levels tended to decrease in several regions, whereas the DA levels and the levels of DA metabolites were unaffected in all regions analyzed. Loss of tyrosine hydroxylase (TH) immunoreactivity in the intranigral injection sites and the presence of TH-immunoreactive pyknotic neurons near the borders of the injection sites were observed following both BOAA and BMAA treatments. Furthermore, substance P-immunoreactive terminals in substantia nigra pars reticulata were also found to have disappeared within the lesioned area following either BOAA or BMAA injections. Incubations with both BOAA and BMAA (10(-5) M) reduced high-affinity [3H]NA uptake in cortical synaptosomes to 69% and 41% of controls, respectively, whereas the striatal high-affinity [3H]DA uptake and the cortical high-affinity [3H]5-HT uptake were unaffected by BOAA or BMAA. The results demonstrate that both BOAA and BMAA can affect central monoamine neurons, although the potency and specificity of these substances on monoamine neurons when administered acutely into cerebral tissue or liquor cerebri seem to be low. However, the in vitro studies indicate selective effects of both compounds on NA neurons in synaptosomal preparations.     

Microscopical investigation of poly(3-hydroxybutyrate) granule formation in Azotobacter vinelandii

Poly(3-hydroxybutyrate) (PHB) granule formation in Azotobacter vinelandii was investigated by laser scanning fluorescence microscopy after staining the cells with Nilered and Baclight. Cells that had been starved for a carbon source for > or =3 days were almost free of PHB granules. Formation of visible PHB granules started within 1-2 h after transfer of the cells to a medium permissive for PHB accumulation. Fluorescent PHB granules at the early stages of formation were exclusively found in the cell periphery of the 2-3 mum ovoid-shaped cells. After 3 h of PHB accumulation or later, PHB granules were also found to be detached from the cell periphery. Our results indicate that PHB granule formation apparently begins at the inner site of the cytoplasmic membrane. This finding is different from previous assumptions that PHB granule formation occurs randomly in the cytoplasm of PHB-accumulating bacteria.     

Production of poly(D-3-hydroxybutyrate) from CO(2), H(2), and O(2) by high cell density autotrophic cultivation of Alcaligenes eutrophus

Hydrogen-oxidizing bacterium, Alcaligenes eutrophus autotrophically produces biodegradable plastic material, poly(D-3-hydroxybutyrate), P(3HB), from carbon dioxide, hydrogen, and oxygen. In autotrophic cultivation of the microorganism, it is essential to eliminate possible occurrence of gas explosions from the fermentation process. We developed a bench-plant scale, recycled-gas, closed-circuit culture system equipped with several safety features to perform autotrophic cultivation of A. eutrophus by maintaining the oxygen concentration in the substrate gas phase below the lower limit for a gas explosion (6.9%). The culture vessel utilized a baskettype agitator, resulting in a K(L) a value of 2970 h(-1). Oxygen gas was also directly fed to the fermentor separately from the other gases. As a result, 91.3 g . dm(-3) of the cells and 61.9 g . dm(-3) of P(3HB) were obtained after 40 h of cultivation under this oxygen-limited condition. The results compared favorably with those reported for mass production of P(3HB) by heterotrophic fermentation. (c) 1995 John Wiley & Sons, Inc.     

Regulation of poly(β-hydroxybutyrate) synthesis in Methylobacterium rhodesianum MB 126 growing on methanol or fructose

The intracellular concentration of CoA metabolites and nucleotides was determined in batch cultures of Methylobacterium rhodesianum grown on methanol and shifted to growth on fructose. The intracellular concentration of CoA decreased from a high value of 0.6 nmol/mg poly(β-hydroxybutyrate)-free bacterial dry mass during growth on methanol to a low value of 0.03 nmol/mg poly(β-hydroxybutyrate)-free bacterial dry mass after a shift to fructose as a carbon source. The levels of NADH, NADPH, and acetyl-CoA were also lower. Under these conditions, acetyl-CoA was metabolized by both citrate synthase and β-ketothiolase, and poly(β-hydroxybutyrate) synthesis and growth occurred simultaneously during growth on fructose. Moreover, the level of ATP was approximately 50% lower during growth on fructose, supporting the hypothesis of a bottleneck in the energy supply during the growth of M. rhodesianum with fructose. Received: 15 July 1997 / Accepted: 10 November 1997