pH regulates cation selectivity of poly-(R)-3-hydroxybutyrate/polyphosphate channels from E. coli in planar lipid bilayers

Poly-(R)-3-hydroxybutyrate/polyphosphate (PHB/polyP) complexes, whether isolated from the plasma membranes of bacteria or prepared from the synthetic polymers, form ion channels in planar lipid bilayers that are highly selective for Ca(2+) over Na(+) at physiological pH. This preference for divalent over monovalent cations is attributed to a high density of negative charge along the polyP backbone and the higher binding energies of divalent cations. Here we modify the charge density of polyP by varying the pH, and observe the effect on cation selectivity. PHB/polyP complexes, isolated from E. coli, were incorporated into planar lipid bilayers, and unitary current-voltage relations were determined as a function of pH. When Ca(2+) was the sole permeant cation, conductance diminished steadily from 97 +/- 6 pS at pH 7.4 to 47 +/- 3 pS at pH 5.5. However, in asymmetric solutions of Ca(2+) and Na(+), there was a moderate increase in conductance from 98 +/- 4 at pH 7.4 to 129 +/- 4 pS at pH 6.5, and a substantially larger increase to 178 +/- 6 pS at pH 5.6, signifying an increase in Na(+) permeability or disorganization of channel structure. Reversal potentials point to a sharp decrease in preference for Ca(2+) over Na(+) over a relatively small decrease in pH. Ca(2+) was strongly favored over Na(+) at physiological pH, but the channels became nonselective near the pK(2) of phosphate (approximately 6.8), and displayed weak selectivity for Na(+) over Ca(2+) at acidic pH. Evidently, PHB/polyP complexes are versatile ion carriers whose selectivity may be modulated by small adjustments of the local pH. The results may be relevant to the physiological function of PHB/polyP channels in bacteria and the role of PHB and polyP in the Streptomyces lividans potassium channel.     

Effect of growth conditions on the molecular weight of poly-3-hydroxybutyrate produced by Azotobacter chroococcum 7B

It has been shown that poly-3-hydroxybutyrate (PHB) of predetermined molecular weight can be obtained by varying the growth conditions of the producer strain, Azotobacter chroococcum 7B: pH, temperature, aeration, presence of sodium acetate as an additional carbon source, or growth on crude complex carbon sources (molasses, vinasse, or starch). High-molecular-weight polymer can be obtained at pH 7.0, optimal for the culture (1485 kDa), temperature 30-37 degrees C (1600-1450 kDa, respectively), and low aeration (2215 kDa). The following factors decrease PHB MW: pH deviation to the acidic (pH 6.0, 476 kDa) or alkaline (pH 8.0, 354 kDa) range or lower temperature (20 degrees C, 897 kDa). Introduction of additional carbon source (sodium acetate) at concentrations in the medium varying from 0 to 5 g/l provides an original method of production of PHB with predetermined MW in a wide range, from 270 to 1515 kDa, with high PHB content in the cell.     

Accumulation of poly-beta-hydroxybutyrate in Nostoc muscorum: regulation by pH, light-dark cycles, N and P status and carbon sources

Accumulation of poly-beta-hydroxybutyrate (PHB) in Nostoc muscorum was studied. Cells harvested at stationary phase of growth depicted maximum accumulation i.e. 8.6% (w/w) of dry cells as compared to lag (4.1%) or logarithmic (6.1%) phases of cultures. In contrast to alkaline pH, acidic pH, continuous illumination and cells grown in presence of combined nitrogen sources, such as NH(4)Cl and KNO(3), were found to affect PHB accumulation negatively. However, P-deficiency and addition of exogenous carbon sources (acetate, glucose, maltose, fructose and ethanol) were found stimulatory for PHB accumulation. In this report PHB accumulation in N. muscorum was boosted up to 35% (w/w) of dry cells when cells supplemented with 0.2% acetate were subjected to dark incubation for 7 days. Further studies are needed at metabolic engineering level or to apply genetic engineering techniques to improve the expression level of PHB photoproduction in cyanobacteria.     

Relationship between succinate transport and production of extracellular poly(3-hydroxybutyrate) depolymerase in Pseudomonas lemoignei

The relationship between extracellular poly(3-hydroxybutyrate) (PHB) depolymerase synthesis and the unusual properties of a succinate uptake system was investigated in Pseudomonas lemoignei. Growth on and uptake of succinate were highly pH dependent, with optima at pH 5.6. Above pH 7, growth on and uptake of succinate were strongly reduced with concomitant derepression of PHB depolymerase synthesis. The specific succinate uptake rates were saturable by high concentrations of succinate, and maximal transport rates of 110 nmol/mg of cell protein per min were determined between pH 5.6 and 6. 8. The apparent KS0.5 values increased with increasing pH from 0.2 mM succinate at pH 5.6 to more than 10 mM succinate at pH 7.6. The uptake of [14C]succinate was strongly inhibited by several monocarboxylates. Dicarboxylates also inhibited the uptake of succinate but only at pH values near the dissociation constant of the second carboxylate function (pKa2). We conclude that the succinate carrier is specific for the monocarboxylate forms of various carboxylic acids and is not able to utilize the dicarboxylic forms. The inability to take up succinate2- accounts for the carbon starvation of P. lemoignei observed during growth on succinate at pH values above 7. As a consequence the bacteria produce high levels of extracellular PHB depolymerase activity in an effort to escape carbon starvation by utilization of PHB hydrolysis products.     

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.     

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

Intracellular degradation of poly(3-hydroxybutyrate) (PHB) in bacteria is not yet clear. The properties of the autodigestion of native PHB granules from Zoogloea 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.     

Isolation of a Gram-positive poly(3-hydroxybutyrate) (PHB)-degrading bacterium from compost, and cloning and characterization of a gene encoding PHB depolymerase of Bacillus megaterium N-18-25-9

A Gram-positive poly(3-hydroxybutyrate) (PHB)-degrading bacterial strain was isolated from compost. This organism, identified as Bacillus megaterium N-18-25-9, produced a clearing zone on opaque NB-PHB agar, indicating the presence of extracellular PHB depolymerase. A PHB depolymerase gene, PhaZ(Bm), of B. megaterium N-18-25-9 was cloned and sequenced, and the recombinant gene product was purified from Escherichia coli. The N-terminal half region of PhaZ(Bm) shared significant homologies with a catalytic domain of other PHB depolymerases. Although the C-terminal half region of PhaZ(Bm) showed no significant similarity with those of other PHB depolymerases, that region was necessary for the PHB depolymerase activity. Therefore, this enzyme's domain structure is unique among extracellular PHB depolymerase domain structures. The addition of PHB to the medium led to a sixfold increase in PhaZ(Bm) mRNA, while the presence of glucose repressed PhaZ(Bm) expression. The maximum activity was observed at pH 9.0 at 65 degrees C.     

Functional evidence for a supramolecular structure for the Streptomyces lividans potassium channel KcsA

Here we present functional evidence for involvement of poly-(R)-3-hydroxybutyrate (PHB) and inorganic polyphosphate (polyP) in ion conduction and selection at the intracellular side of the Streptomyces lividans potassium channel, KcsA. At < or = 25 degrees C, KcsA forms channels in planar bilayers that display signal characteristics of PHB/polyP channels at the intracellular side; i.e., a preference for divalent Mg(2+) cations at pH 7.2, and a preference for monovalent K+ cations at pH 6.8. Between 25 and 26 degrees C, KcsA undergoes a transition to a new conformation in which the channel exhibits high selectivity for K+, regardless of solution pH. This suggests that basic residues of the C-terminal polypeptides have moved closer to the polyP end unit, reducing its negative charge. The data support a supramolecular structure for KcsA in which influx of ions is prevented by the selectivity pore, whereas efflux of K+ is governed by a conductive core of PHB/polyP in partnership with the C-terminal polypeptide strands.     

Preparation,statistical optimization and characterization of poly(3-hydroxybutyrate) fermented by Cupriavidus necator utilizing various hydrolysates of alligator weed (Alternanthera philoxeroides) as a sole carbon source

Alligator weed (Alternanthera philoxeroides) is a stoloniferous, amphibious and perennial herb which has invaded many parts of the world and led to serious environmental and ecological problems. In order to exploit cheap carbon source for poly(3-hydroxybutyrate) (PHB) production, alligator weed hydrolysates were prepared by acid and enzyme treatment and used for PHB production via Cupriavidus necator. The bacterium utilized alligator weed enzymatic hydrolysate and produced the PHB concentration of 3.8 ± 0.2 g/L at the conditions of pH 7.0, 27.5°C, 1.5 g/L of nitrogen source, and 25 g/L of carbon source, this exceeded the value of 2.1 ± 0.1 g/L from acid hydrolysate media at the same conditions. In order to obtain the optimum conditions of PHB production, response surface methodology was employed which improved PHB content. The optimum conditions for PHB production are as follows: carbon source, 34 g/L; nitrogen source, 2 g/L; pH, 7; temperature, 28°C. After 72 hr of incubation, the bacterium produced 8.5 g/L of dry cell weight and 4.8 g/L of PHB. The PHB was subjected to Fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC), and Molecular weight analysis and found the melting temperature, number average molecular mass, and polydispersity were 168.20°C, 185 kDa, and 2.1, respectively.     

Relationship between Succinate Transport and Production of Extracellular Poly(3-Hydroxybutyrate) Depolymerase in Pseudomonas lemoignei

The relationship between extracellular poly(3-hydroxybutyrate) (PHB) depolymerase synthesis and the unusual properties of a succinate uptake system was investigated in Pseudomonas lemoignei. Growth on and uptake of succinate were highly pH dependent, with optima at pH 5.6. Above pH 7, growth on and uptake of succinate were strongly reduced with concomitant derepression of PHB depolymerase synthesis. The specific succinate uptake rates were saturable by high concentrations of succinate, and maximal transport rates of 110 nmol/mg of cell protein per min were determined between pH 5.6 and 6.8. The apparent K_S0.5 values increased with increasing pH from 0.2 mM succinate at pH 5.6 to more than 10 mM succinate at pH 7.6. The uptake of [¹⁴C]succinate was strongly inhibited by several monocarboxylates. Dicarboxylates also inhibited the uptake of succinate but only at pH values near the dissociation constant of the second carboxylate function (pK_a2). We conclude that the succinate carrier is specific for the monocarboxylate forms of various carboxylic acids and is not able to utilize the dicarboxylic forms. The inability to take up succinate²⁻ accounts for the carbon starvation of P. lemoignei observed during growth on succinate at pH values above 7. As a consequence the bacteria produce high levels of extracellular PHB depolymerase activity in an effort to escape carbon starvation by utilization of PHB hydrolysis products.     

Effect of growth conditions on the molecular weight of poly-3-hydroxybutyrate produced by <Emphasis Type="Italic">Azotobacter chroococcum</Emphasis> 7B

It has been shown that poly-3-hydroxybutyrate (PHB) of predetermined molecular weight can be obtained by varying the growth conditions of the producer strain, Azotobacter chroococcum 7B: pH, temperature, aeration, presence of sodium acetate as an additional carbon source, or growth on crude complex carbon sources (molasses, vinasse, or starch). High-molecular-weight polymer can be obtained at pH 7.0, optimal for the culture (1485 kDa), temperature 30–37°C (1600–1450 kDa, respectively), and low aeration (2215 kDa). The following factors decrease PHB MW: pH deviation to the acidic (pH 6.0, 476 kDa) or alkaline (pH 8.0, 354 kDa) range or lower temperature (20°C, 897 kDa). Introduction of additional carbon source (sodium acetate) at concentrations in the medium varying from 0 to 5 g/l provides an original method of production of PHB with predetermined MW in a wide range, from 270 to 1515 kDa, with high PHB content in the cell.     

An extracellular poly (3-hydroxybutyrate) depolymerase from Penicillium sp. DS9713a-01

Summary Penicillium sp. DS9713a-01 was obtained by ultraviolet (u.v.) light mutagenesis from the Penicillium sp. DS9713a which can degrade poly (3-hydroxybutyrate) (PHB). The enzymatic activity of DS9713a-01 was 97% higher than that of the wild-type strain. The DS9713a-01 mutant could completely degrade PHB films in 5 days; however, the wild-type strain achieved only 61% at the same time. The extracellular PHB depolymerase was purified from the culture medium containing PHB as the sole carbon source by filtration, ammonium sulfate precipitation and chromatography on Sepharose CL-6B. The molecular weight of the PHB depolymerase was about 15.1kDa determined by SDS-polyacrylamide gel electrophoresis. The optimum activity of the PHB depolymerase was observed at pH 8.6 and 50 °C. The enzyme was stable at temperatures below 37 °C and in the pH range from 8.0 to 9.2. The activity of PHB depolymerase could be activated or inhibited by some metal ions. The apparent K _m value was 0.164 mg ml⁻¹. Mass spectrometric analysis of the water-soluble products after enzymatic degradation revealed that the primary product was the monomer, 3-hydroxybutyric acid.     

Accumulation of poly-(hydroxybutyrate) by a non-sulfur photosynthetic bacterium,Rhodobacter sphaeroides RV at different pH

Summary Effect of pH of culture media on intracellular accumulation of poly-(hydroxybutyrate) (PHB) by a non-sulfur photosynthetic bacterium, Rhodobacter sphaeroides strain RV was studied in pH-stat cultures. Sub-optimal pH for growth, 8.0 or 8.5 gave the higher content of PHB rather than optimal pH 7.5 for growth. These results show that growth and PHB accumulation of the bacteria can be controlled by pH of culture media.     

Molecular mass of poly[(R )-3-hydroxybutyric acid] produced in a recombinant Escherichia coli

 Poly[(R)-3-hydroxybutyric acid] (PHB) was produced at 37 °C by a recombinant Escherichia coli harboring the Alcaligenes eutrophus biosynthesis phbCAB genes in Luria-Bertani media containing glucose at 10–30 g/l at different pH values and the time-dependent changes in the molecular mass of PHB were studied. PHB polymers accumulated within cells while glucose was present in the medium. The number-average molecular mass of PHB decreased with time during the course of PHB accumulation, and the values for PHB were markedly dependent on the cultivation conditions of the E. coli, ranging from 0.5 MDa to 20 MDa. Under specific conditions (pH 6.0), E. coli produced PHB with an extremely high molecular mass (20 MDa). It has been suggested that a chain-transfer agent is generated in E. coli cells during the accumulation of PHB. Received: 18 July 1996 / Received revision: 4 November 1996 / Accepted: 4 November 1996     

Distribution and Selection of Poly-3-Hydroxybutyrate Production Capacity in Methanotrophic Proteobacteria

Methanotrophs are known to produce poly-3-hydroxybutyrate (PHB), but there is conflicting evidence in the literature as to which genera produce the polymer. We screened type I and II proteobacterial methanotrophs that use the ribulose monophosphate and serine pathways for carbon assimilation, respectively, for both phaC, which encodes for PHB synthase, and the ability to produce PHB under nitrogen-limited conditions. Twelve strains from six different genera were evaluated. All type I strains tested negative for phaC and PHB production; all Type II strains tested positive for phaC and PHB production. In order to identify conditions that favor PHB production, we also evaluated a range of selection conditions using a diverse activated sludge inoculum. Use of medium typically recommended for methanotroph enrichment led to enrichments dominated by type I methanotrophs. Conditions that were selected for enrichments dominated by PHB-producing Type II methanotrophs were: (1) use of nitrogen gas as the sole nitrogen source in the absence of copper, (2) use of a dilute mineral salts media in the absence of copper, and (3) use of media prepared at pH values of 4–5.     

Nutritional flexibility of oligotrophic and copiotrophic Antarctic bacteria with respect to organic substrates

Abstract Alcaligenes eutrophus can accumulate poly-3-hydroxybutyrate (PHB) or polyhydroxyalkanoate (PHA) containing only 3-hydroxybutyrate (HB) and 3-hydroxyvalerate (HV) units. Granule-associated PHB-synthase was active with d (−)-3-hydroxybutyryl-CoA and d (−)-3-hydroxyvaleryl-CoA of the range of d (−)- and l (+)-3-hydroxyacyl-CoA substrates tested (C₄–C₁₀). In carbon-limited cultures, PHB-synthase was predominantly soluble, becoming granule-associated on transition to nitrogen limitation. Granule-associated PHB-synthase increased in activity at least up to pH 10.0 and K _m values of 0.68 mM and 1.63 mM were determined for the C₄ and C₅ substrates, respectively, at pH 8.5. The soluble PHB-synthase, which was unstable, showed equal activity in the range pH 8.0–10.0, had a K _m value for d (−)-3-hydroxybutyryl-CoA of 0.72 mM and an M _r of 160,000. PHB does not measurably turn over under steady-state polymer-accumulating conditions.     

Metabolic and kinetic analysis of poly(3-hydroxybutyrate) production by recombinant Escherichia coli

A quantitatively repeatable protocol was developed for poly(3-hydroxybutyrate) (PHB) production by Escherichia coli XL1-Blue (pSYL107). Two constant-glucose fed-batch fermentations of duration 25 h were carried out in a 5-L bioreactor, with the measured oxygen volumetric mass-transfer coefficient (k(L)a) held constant at 1.1 min(-1). All major consumption and production rates were quantified. The intracellular concentration profiles of acetyl-CoA (300 to 600 microg x g RCM(-1)) and 3-hydroxybutyryl-CoA (20 to 40 microg x g RCM(-1)) were measured, which is the first time this has been performed for E. coli during PHB production. The kinetics of PHB production were examined and likely ranges were established for polyhydroxyalkanoate (PHA) enzyme activity and the concentration of pathway metabolites. These measured and estimated values are quite similar to the available literature estimates for the native PHB producer Ralstonia eutropha. Metabolic control analysis performed on the PHB metabolic pathway showed that the PHB flux was highly sensitive to acetyl-CoA/CoA ratio (response coefficient 0.8), total acetyl-CoA + CoA concentration (response coefficient 0.7), and pH (response coefficient -1.25). It was less sensitive (response coefficient 0.25) to NADPH/NADP ratio. NADP(H) concentration (NADPH + NADP) had a negligible effect. No single enzyme had a dominant flux control coefficient under the experimental conditions examined (0.6, 0.25, and 0.15 for 3-ketoacyl-CoA reductase, PHA synthase, and 3-ketothiolase, respectively). In conjunction with metabolic flux analysis, kinetic analysis was used to provide a metabolic explanation for the observed fermentation profile. In particular, the rapid onset of PHB production was shown to be caused by oxygen limitation, which initiated a cascade of secondary metabolic events, including cessation of TCA cycle flux and an increase in acetyl-CoA/CoA ratio.     

Influence of Ammonium Salts and Cane Molasses on Growth of Alcaligenes eutrophus and Production of Polyhydroxybutyrate

The production of polyhydroxybutyrate (PHB) by Alcaligenes eutrophus DSM 545 was studied in a synthetic medium with 3% glucose at pH 7.0 supplemented with several ammonium substrates and cane molasses. Growth was measured by dry cell weight, and the PHB content was measured by gas chromatography. The effects of ammonium sources such as sulfate, nitrate, phosphate, and chloride salts and those of different ammonium sulfate concentrations were evaluated. The best growth and PHB production were obtained with ammonium sulfate; however, NH(inf4)(sup+) concentrations between 0.5 and 1.5 g/liter showed no significant difference. Ammonium sulfate was therefore used as the sole source of NH(inf4)(sup+) for experiments with cane molasses as the growth activator. Optimal growth and PHB production were obtained with 0.3% molasses. However, the yields of biomass (39 to 48%) and PHB (17 to 26%) varied significantly among the different ammonium substrates and cane molasses concentrations.     

High level production of supra molecular weight poly (3-hydroxybutyrate) by metabolically engineered<Emphasis Type="Italic">Escherichia coli</Emphasis>

The supra molecular weight poly ([R]-3-hydroxybutyrate) (PHB), having a molecular weight greater than 2 million Da, has recently been found to possess improved mechanical properties compared with the normal molecular weight PHB, which has a molecular weight of less than 1 million Da. However, applications for this PHB have been hampered due to the difficulty of its production. Reported here, is the development of a new metabolically engineeredEscherichia coli strain and its fermentation for high level production of supra molecular weight PHB. RecombinantE. coli strains, harboring plasmids of different copy numbers containing theAlcaligenes latus PHB biosynthesis genes, were cultured and the molecular weights of the accumulated PHB were compared. When the recombinantE. coli XL 1-Blue, harboring a medium-copy-number pJC2 containing theA. latus PHB biosynthesis genes, was cultivated by fed-batch culture at pH 6.0, supra molecular weight PHB could be produced at up to 89.8 g/L with a productivity of 2.07 g PHB/L-h. The molecular weight of PHB obtained under these conditions was as high as 22 MDa, exceeding by an order of magnitude the molecular weight of PHB typically produced inRalstonia eutropha or recombinantE. coli     

Proteome analysis of metabolically engineered Escherichia coli producing Poly(3-hydroxybutyrate)

Recombinant Escherichia coli strains harboring heterologous polyhydroxyalkanoate (PHA) biosynthesis genes were shown to accumulate unusually large amounts of PHA. In the present study, integrated cellular responses of metabolically engineered E. coli to the accumulation of poly(3-hydroxybutyrate) (PHB) in the early stationary phase were analyzed at the protein level by two-dimensional gel electrophoresis. Out of 20 proteins showing altered expression levels with the accumulation of PHB, 13 proteins were identified with the aid of mass spectrometry. Three heat shock proteins, GroEL, GroES, and DnaK, were significantly up-regulated in PHB-accumulating cells. Proteins which play essential roles in protein biosynthesis were unfavorably influenced by the accumulation of PHB. Cellular demand for the large amount of acetyl coenzyme A and NADPH for the PHB biosynthesis resulted in the increased synthesis of two enzymes of the glycolytic pathway and one enzyme of the Entner-Doudoroff pathway. The expression of the yfiD gene encoding a 14.3-kDa protein, which is known to be produced at low pH, was greatly induced with the accumulation of PHB. Therefore, it could be concluded that the accumulation of PHB in E. coli acted as a stress on the cells, which reduced the cells' ability to synthesize proteins and induced the expression of various protective proteins.