Bacterial synthesis of polyhydroxyalkanoates containing aromatic and aliphatic monomers by Pseudomonas putida CA-3

Pseudomonas putida CA-3 has the ability to accumulate to high levels unique polyhydroxyalkanoate (PHA) heteropolymers composed of aromatic and aliphatic monomers. The majority of monomers are aromatic making up 98% of the polymer. (R)-3-hydroxyphenylvalerate and (R)-3-hydroxyphenylhexanoate are the most abundant monomers found in polymers accumulated from phenylalkanoic acids with an uneven and even number of carbons on the acyl side chain respectively. PHAs accumulated from phenylvaleric and phenylhexanoic acid were partially crystalline while all other PHAs were amorphous. Significant differences in the yield and PHA content of the cells occurred when different phenylalkanoic acids were supplied as growth substrates. Increasing the initial concentration of the growth substrate increased both the PHA content of the cells and the overall yield (g PHA/g carbon supplied) of PHA accumulated by P. putida CA-3 cells. The highest PHA content (% cell dry wt.) from an aromatic carbon source was 59% when 15mM phenylvaleric acid was supplied as the sole source of carbon and energy. This corresponded to a maximum PHA yield of 0.42 g PHA/g carbon supplied. In and attempt to increase the level of PHA accumulated from related growth substrates acrylic acid was added to the growth medium. However, the addition of various concentrations of acrylic acid to the growth medium had either no effect or decreased the PHA content of the cell accumulated from phenylalkanoic acids by P. putida CA-3.     

真氧产碱杆菌在双营养（碳、氮）限制区内合成聚羟基烷酸酯

研究了真氧产碱杆菌以混合有机酸为碳源,硫酸铵为氮源,在双营养（碳、氮）限制区内聚羟基烷酸酯的生物合成。结果表明：双营养限制区的长度与聚羟基烷酸酯的产量呈正相关。同时,在对两种不同的双营养限制区实现方式进行比较后发现,首先限制碳源的双营养限制方式比首先限制氮源的双营养限制方式更有利于聚羟基烷酸酯的合成;在这两种不同营养限制方式下,PHAs的最高产量分别为3.72 g/L和2.55 g/L。     

Biodegradation of Poly-(beta)-Hydroxyalkanoates in a Lake Sediment Sample Increases Bacterial Sulfate Reduction

The addition of a poly-(beta)-hydroxyalkanoate (PHA) to incubation bottles containing anaerobic sediment from Lake Ciso (Banyoles, Catalonia, northeast Spain) was correlated with an increase in sulfide production. The concentration of PHA diminished to 51 to 99% of the initial amount. Sodium molybdate (1 mM) decreased the rate of PHA degradation and inhibited sulfide production, thus indicating that PHAs serve as carbon and electron sources for sulfate reduction.     

Polyhydroxyalkanoate (PHA) biosynthesis from structurally unrelated carbon sources by a newly characterized Bacillus spp

A newly acquired polyhydroxyalkanoate (PHA) producing Bacillus spp. was identified to be a strain of Bacillus cereus using a range of microbiological and molecular techniques. This strain, named B. cereus SPV, was found to be capable of using a wide range of carbon sources including glucose, fructose, sucrose, various fatty acids and gluconate for the production of PHAs, an advantage for the commercial production of the polymers. The media used for the polymer production was novel in the context of the genus Bacillus. The PHA, once produced, was found to remain at a constant maximal concentration, without any degradation, a great advantage for the commercial production of the PHAs. This particular strain of Bacillus spp. was able to synthesize various PHAs with 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV) and 4-hydroxybutyrate (4HB)-like monomer units from structurally unrelated carbon sources such as fructose, sucrose and gluconate. This is the first report of the incorporation of a 4HB related monomer containing PHA by the genus Bacillus and from structurally unrelated carbon sources. The PHAs isolated had molecular weights ranging between (0.4 and 0.8) x 10(6) and low polydispersity index values (M(W)/M(N)) ranging from 2.6 to 3.4.     

Formation of short chain length/medium chain length polyhydroxyalkanoate copolymers by fatty acid beta-oxidation inhibited Ralstonia eutropha

Ralstonia eutropha has been considered as a bacterium, incorporating hydroxyalkanoates of less than six carbons only into polyhydroxyalkanoates (PHAs). Cells of the wild type cultivated with sodium octanoate as the carbon source in the presence of the fatty acid beta-oxidation inhibitor sodium acrylate synthesized PHAs composed of the medium chain length hydroxyalkanoates (3HA(MCL)) 3-hydroxyhexanoate (3HHx) and 3-hydroxyoctanoate (3HO) as well as of 3-hydroxybutyrate and 3-hydroxyproprionate as revealed by gas chromatography, (1)H NMR spectroscopy, and mass spectroscopy. The characterization of the polymer as a tetrapolymer was confirmed by differential solvent extraction and measurement of melting and glass transition temperature depression in the purified polymer compared to PHB. These data suggested that the R. eutropha PHA synthase is capable of incorporating longer chain substrates than suggested by previous in vitro studies. Furthermore, expression of the class II PHA synthase gene phaC1 from P. aeruginosa in R. eutropha resulted in the accumulation of PHAs consisting of 3HA(MCL) contributing about 3-5% to cellular dry weight. These PHAs were composed of nearly equal molar fractions of 3HO and 3-hydroxydecanoate (3HD) with traces of 3HHx. These data indicated that 3HA(MCL)-CoA thioesters were diverted from the fatty acid beta-oxidation pathway towards PHA biosynthesis in recombinant R. eutropha.     

13C nuclear magnetic resonance studies of Pseudomonas putida fatty acid metabolic routes involved in poly(3-hydroxyalkanoate) synthesis.

The formation of poly(3-hydroxyalkanoates) (PHAs) in Pseudomonas putida KT2442 from various carbon sources was studied by 13C nuclear magnetic resonance spectroscopy, gas chromatography, and gas chromatography-mass spectroscopy. By using [1-13C]decanoate, the relation between beta-oxidation and PHA formation was confirmed. The labeling pattern in PHAs synthesized from [1-13C]acetate corresponded to the formation of PHAs via de novo fatty acid biosynthesis. Studies with specific inhibitors of the fatty acid metabolic pathways demonstrated that beta-oxidation and de novo fatty acid biosynthesis function independently in PHA formation. Analysis of PHAs derived from [1-13C]hexanoate showed that both fatty acid metabolic routes can function simultaneously in the synthesis of PHA. Furthermore, evidence is presented that during growth on medium-chain-length fatty acids, PHA precursors can be generated by elongation of these fatty acids with an acetyl coenzyme A molecule, presumably by a reverse action of 3-ketothiolase.     

Biosynthesis and structural characterization of medium-chain-length poly(3-hydroxyalkanoates) produced by Pseudomonas aeruginosa from fatty acids

In this study, we investigated the ability of Pseudomonas aeruginosa ATCC 27853 to grow and synthesize poly(3-hydroxyalkanoates) (PHAs) from saturated fatty acids with an even number of carbon atoms, from eight to 22, and from oleic acid. In a non-limiting medium, all carbon sources but docosanoic acid supported cell growth and PHA production, with eicosanoic acid giving the highest yield. In magnesium-limiting conditions, higher yields were obtained from sources with up to 16 carbon atoms. Composition was estimated by gas chromatography of methanolyzed samples and (13)C nuclear magnetic resonance. The 3-hydroxyalkanoate units extended from hexanoate to tetradecanoate or tetradecenoate, with octanoate and decanoate as the predominant components. Weight average molecular weights ranged from 78,000 to 316,000. Fast atom bombardment mass spectrometry of partially pyrolyzed samples, coupled to statistical analysis, showed that these PHAs are random copolymers.     

Production of medium-chain-length polyhydroxyalkanoates by high-cell-density cultivation of Pseudomonas putida under phosphorus limitation

High-cell-density fed-batch cultures of Pseudomonas putida were carried out for the production of medium-chain-length polyhydroxyalkanoates (PHAs) using oleic acid as a carbon source. By employing an optimal feeding strategy without the limitation of any nutrient, a high cell concentration of 173 g/L was achieved, but the PHA concentration and PHA content were only 32.3 g/L and 18.7 wt%, respectively. To increase the PHA concentration and content, phosphorus limitation was applied during fed-bath culture by reducing the initial KH(2)PO(4) concentration. When the initial KH(2)PO(4) concentration was reduced to 4 g/L, cell concentration, PHA concentration, and PHA content obtained in 38 h were 141 g/L, 72. 6 g/L, and 51.4 wt%, respectively, resulting in a high productivity of 1.91 g PHA/L per hour.     

Structure and polymer form of poly-3-hydroxyalkanoates produced by Pseudomonas oleovorans grown with mixture of sodium octanoate/undecylenic acid and sodium octanoate/5-phenylvaleric acid

PHAs (poly-3-hydroxyalkanoates) obtained by Pseudomonas oleovorans grown with mixed carbon sources were investigated. Mixed carbon sources were sodium octanoate/undecylenic acid and sodium octanoate/5-phenylvaleric acid. Effect of carbon source in pre-culture on PHAs structure was investigated. Main fermentation was conducted with mixture of sodium octanoate/undecylenic acid, and PHA contained both saturated and unsaturated units. When more undecylenic acid was used in the medium, the ratio of unsaturated unit increased and the T(g) of the products also changed. The PHA grown with mixture of sodium octanoate and undecylenic acid was a random copolymer, which was determined by DSC analysis. Using mixed carbon sources of sodium octanoate and 5-phenylvaleric acid, highest dry cell weight and PHA concentration were obtained when 0.02g or 0.04g of 5-phenylvaleric acid were added in 50mL medium. Cultured with sodium octanoate and 5-phenylvaleric acid, PHA containing HO (3-hydroxyoctanoate) unit and HPV (3-hydroxy-5-phenylvalerate) unit was produced. T(g) of the products fell between those of pure PHO and pure PHPV. By means of DSC analysis and fractionation method, the PHA obtained was regarded as a random copolymer.     

Mobilization of cadmium from Festuca ovina roots and its simultaneous immobilization by soil in a root-soil-extractant system (in vitro test)

Mobilization of cadmium accumulated in Festuca ovina L. roots and simultaneous immobilization of this metal by soil were studied in three chambers connected into one system containing: (1) roots in an extractant solution, (2) soil in an extractant solution, and (3) extractant solution alone. Six extractants sterilized by filtration were used: 0.1 M NaNO₃ (NA), 0.1 mM desferrioxamine B (NA + DFOB) and 1 mM citric acid (NA + CA) in NA, and a water extract of soil (SE) supplemented with the same compounds. SE mobilized 53% of the Cd introduced to the system with roots. The addition of DFOB or CA to SE increased Cd extraction from roots by 17%, while the same compounds introduced to NA did not change mobilization of Cd (60% efficiency). Regardless of the extractant used, mobilization of Cd from roots was about 25% lower when extraction was done in a control system without soil. The metal released from roots was gradually immobilized by the soils loaded into all systems during a 4-day incubation. Sequential extractions of Cd from the soils showed that the metal released from roots with NA was stabilized mainly by soil Mn and Fe oxides, while that released with SE was stabilized by soil organic matter.     

利用两种工程菌共培养生产聚羟基烷酸的研究

方法:主要是将携带生产P(3HB)基因的工程菌E.coliXL1-Blue(pKSABC)和携带4-羟基丁酸辅酶A转移酶和PHA合成酶基因的工程菌E.coliXL1-Blue(pKSSE5.3)共培养,以葡萄糖为唯一碳源来生产含有3HB和4HB的聚合物的研究。结果:培养48h后,结果表明,两种工程菌共培养能够获得韧性更强和强度更大的PHA,PHA的积累量占总生物量的44.4%。     

Redox Modulation of N-Methyl-D-Aspartate-Stimulated Neurotransmitter Release from Rat Brain Slices

Rat brain cortical slices released tritiated norepinephrine ([3H]NA) during a 2-min stimulation with N-methyl-D-aspartate (NMDA). Dithiothreitol (DTT; 0.1-5 mM), present for 6 min prior to stimulation, dose-dependently increased the release of [3H]NA from cortical slices stimulated with a maximally effective concentration of NMDA (500 microM). Similar results were observed for [3H]NA release from hippocampal slices and tritiated and endogenous dopamine release from striatal slices. DTT treatment also markedly shifted the dose-response curve of NMDA to the left. Cortical slices released approximately the same amount of [3H]NA with 10 microM NMDA following DTT treatment (about 5%) as non-DTT-treated control slices did with 500 microM NMDA. The effects of DTT were fully reversed by subsequent treatment with 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB; 0.5 mM). DTT treatment did not significantly alter the ability of magnesium (1.3 mM) or the polyamine antagonist arcaine to block the NMDA-stimulated release of [3H]NA. In contrast, DTT treatment significantly attenuated the antagonist effects of the competitive glycine antagonist, 7-chlorokynurenic acid, and the competitive NMDA antagonist, 2-aminophosphonopentanoic acid. These results suggest that oxidation and reduction of disulfide bonds located within the NMDA receptor complex might regulate the activation of the NMDA receptor. This could have important consequences in vivo if endogenous oxidizing/reducing systems are found to have similar effects on NMDA-stimulated responses.     

Production of polyhydroxyalkanoates from agricultural waste and surplus materials

To be competitive with common plastics, the production costs of polyhydroxyalkanoates (PHAs) have to be minimized. Biotechnological polymer production occurs in aerobic processes; therefore, only about 50% of the main carbon sources and even a lower percentage of the precursors used for production of co-polyesters end up in the products wanted. A second cost factor in normally phosphate-limited production processes for PHAs is the costs for complex nitrogen sources. Both cheap carbon sources and cheap nitrogen sources are available from agricultural waste and surplus materials and make a substantial contribution for minimizing PHA production costs. In this study, fermentations for PHA production were carried out in laboratory-scale bioreactors on hydrolyzed whey permeate and glycerol liquid phase from the biodiesel production using a highly osmophilic organism. Without any precursor, the organism produced a poly[3(hydroxybutyrate-co-hydroxyvalerate)] copolyester on both carbon sources. During the accumulation phases, a constant 3-hydroxyvalerate content of 8-10% was obtained at a total PHA concentration of 5.5 g/L (on hydrolyzed whey permeate) and 16.2 g/L (glycerol liquid phase). In an additional fermentation, an expensive nitrogen source was substituted by meat and bone meal beside the glycerol liquid phase as a carbon source, resulting in a final PHA concentration of 5.9 g/L.     

Neomycin inhibits K+- and veratridine-stimulated noradrenaline release in rat brain slices and rat brain synaptosomes

The possible involvement of phosphoinositides' turnover in the process of neurotransmitter release in the central nervous system (CNS) was studied using rat brain slices and synaptosomes. A depolarizing concentration of potassium chloride (25 mM) induces an 8.6 +/- 0.4% increase of [3H]noradrenaline [( 3H]NA) fractional release in cerebral cortical slices above spontaneous release, and 15 mM KCl induces a 3-fold increase of [3H]NA release in rat brain synaptosomes. Neomycin, an aminoglycoside which binds phosphoinositides, inhibits the potassium-induced release in cortical slices with an IC50 = 0.5 +/- 0.07 mM and with IC50 = 0.2 +/- 0.03 mM in synaptosomes. Veratridine, a veratrum alkaloid which increases membrane permeability to sodium ions and causes depolarization of neuronal cells, induces a net 13.4 +/- 0.3% increase of [3H]NA fractional release above spontaneous release in cortical slices. In analogy to K+ stimulation, neomycin inhibits the veratridine-stimulated release in cortical slices with an IC50 = 0.65 +/- 0.1 mM. It appears that the recycling of phosphoinositides, which is necessary for Ca2+ mobilization, participates in the Ca2+-dependent induced neurotransmitter release in the central nervous system.     

Effects of noradrenaline and nicotinic acid on plasma free fatty acids and oxygen consumption in cold-adapted rats.

A 3-h noradrenaline (NA) infusion (1.5 microgram kg-1 min-1) produced a sustained enhanced oxygen consumption (O2 cons.) in cold-adapted rats. Plasma free fatty acid (FFA) levels were elevated by NA in control and in cold-adapted rats, but to lesser extent in cold-adapted rats; the increase was maintained at a plateau in both groups during the entire period of NA infusion. A 1-h nicotinic acid (Nic A) infusion (1.5 mg kg-1 min-1) added to the NA infusion inhibited the calorigenic response to NA in cold-adapted rats and reduced the elevated plasma FFA concentration in control and in cold-adapted rats to values below basal levels. However, when the Nic A infusion was stopped, the O2 cons. was increased again in cold-adapted rats by the uninterrupted NA infusion, without the simultaneous increase of the plasma FFA concentration; the plasma FFA concentration was maintained in cold-adapted rats below basal values and merely brought back to basal levels in control rats. From these results, it is suggested that plasma FFA are not an essential substrate to the calorigenic response to NA observed in cold-adapted rats, as 85% of the response can occur when the plasma FFA concentration is very low.     

High-performance amperometric biosensors and biofuel cell based on chitosan-strengthened cast thin films of chemically synthesized catecholamine polymers with glucose oxidase effectively entrapped

Rapid oxidation of dopamine (DA) or L-noradrenaline (NA) by K(3)Fe(CN)(6) yields poly(DA) (PDA(C)) or poly(NA) (PNA(C)) with glucose oxidase (GOx) effectively entrapped, and such an enzyme-entrapped catecholamine polymer is cast on an Au electrode followed by chitosan (CS) strengthening for biosensing and fabrication of a biofuel cell (BFC). The optimized glucose biosensor of CS/PDA(C)-GOx/Au displays an extremely high sensitivity up to 135 μA mM(-1) cm(-2), a very low limit of detection of 0.07 μM, a response time of <3 s, good suppression of interferents, striking thermostability (lifetime of 3 weeks at 60°C and over 2 months at 30°C), and high resistance to urea denaturation. The biosensor also works well in the second generation biosensing mode with p-benzoquinone (BQ) or ferrocene monocarboxylic acid (Fc) as an artificial mediator, with greatly broadened linear detection ranges (2.0 μM-48.0 mM for BQ and 2.0 μM-16.0 mM for Fc) and up to mA cm(-2)-scale glucose-saturated current density. The good permeability of artificial mediators across the enzyme film enables the quantification of the surface concentration of immobilized GOx on the basis of a reported kinetic model, and UV-Vis spectrophotometry is used to measure the enzymatic activity, revealing high enzymatic activity/load at CS/PDA(C)-GOx/Au. A BFC is also successfully fabricated with a bioanode of CS/PDA(C)-GOx/Au in phosphate buffer solution containing 100 mM glucose and 4.0 mM BQ and a carbon cathode in Nafion-membrane-isolated acidic KMnO(4), and its maximum power density of 1.62 mW cm(-2) is superior to those of most BFC hitherto reported.     

A thermostable beta-ketothiolase of polyhydroxyalkanoates (PHAs) in Thermus thermophilus: purification and biochemical properties

Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoates (HAs) synthesised by numerous bacteria as intracellular carbon and energy storage compounds which accumulate as granules in the cytoplasm of the cells. The biosynthesis of PHAs, in the thermophilic bacterium T. thermophilus grown in a mineral medium supplemented with sodium gluconate as sole carbon source has been recently reported. Here, we report the purification at apparent homogeneity of a beta-ketoacyl-CoA thiolase from T. thermophilus, the first enzyme of the most common biosynthetic pathway for PHAs. B-Ketoacyl-CoA thiolase appeared as a single band of 45.5-kDa molecular mass on SDS/PAGE. The enzyme was purified 390-fold with 7% recovery. The native enzyme is a multimeric protein of a molecular mass of approximately of 182 kDa consisting of four identical subunits of 45.5 kDa, as identified by an in situ renaturation experiment on SDS-PAGE. The enzyme exhibited an optimal pH of approximately 8.0 and highest activity at 65 degrees C for both direction of the reaction. The thiolysis reaction showed a substrate inhibition at high concentrations; when one of the substrates (acetoacetyl CoA or CoA) is varied, while the concentrations of the second substrates (CoA or acetoacetyl CoA respectively) remain constant. The initial velocity kinetics showed a pattern of a family of parallel lines, which is in accordance with a ping-pong mechanism. beta-Ketothiolase had a relative low Km of 0.25 mM for acetyl-CoA and 11 microM and 25 microM for CoA and acetoacetyl-CoA, respectively. The enzyme was inhibited by treatment with 1 mM N-ethylmaleimide either in the presence or in the absence of 0.5 mM of acetyl-CoA suggesting that possibly a cysteine is located at/or near the active site of beta-ketothiolase.     

Synthesis and characterization of poly(3-hydroxyalkanoates) from Brassica carinata oil with high content of erucic acid and from very long chain fatty acids

Pseudomonas aeruginosa produced medium chain length poly(3-hydroxyalkanoates) (mcl-PHAs) when grown on substrates containing very long chain fatty acids (VLCFA, C>20). Looking for low cost carbon sources, we tested Brassica carinata oil (erucic acid content 35-48%) as an intact triglyceride containing VLCFA. Oleic (C18:1), erucic (C22:1), and nervonic (C24:1) acids were also employed for mcl-PHA production as model substrates. The polymers obtained were analyzed by GC of methanolyzed samples, GPC, 1H and 13C NMR, ESI MS of partially pyrolyzed samples, and DSC. The repeating units of such polymers were saturated and unsaturated, with a higher content of the latter in the case of the PHA obtained from B. carinata oil. Statistical analysis of the ion intensity in the ESI mass spectra showed that the PHAs from pure fatty acids are random copolymers, while the PHA from B. carinata oil is either a pure polymer or a mixture of polymers. Weight-average molecular weight varied from ca. 56,000 g/mol for the PHA from B. carinata oil and oleic acid, to about 120,000 g/mol for those from erucic and nervonic acids. The PHAs from erucic and nervonic acids were partially crystalline, with rubbery characteristics and a melting point (Tm) of 50°C, while the PHAs from oleic acid and from B. carinata oil afforded totally amorphous materials, with glass transition temperatures (Tg) of -52°C and -47°C, respectively.     

主要营养成分对悬浮培养玫瑰茄细胞生长和花青素合成的影响

本文研究了培养基中碳源和氮源变化对悬浮培养玫瑰茄细胞生长和花青素合成的影响。在８种不同的碳源中,麦芽糖有利于花青素的积累,而蔗糖和葡萄糖适合细胞生长,并有较高的花青素产率。在１％～１０％蔗糖浓度范围内,４％浓度下细胞生长和花青素产率最高,而６％浓度下细胞花青素含量最高,高渗环境较有利于细胞花青素的积累。１３５ｍＭ的氮源总量已足够维持玫瑰茄细胞生长和花青素合成,氮源总量增加对细胞代谢有抑制作用。ＮＨ＋４对细胞有显著抑制作用。总量１３５ｍＭ,ＮＯ－３与ＮＨ＋４比例２５∶２和２３∶４时细胞生长和花青素合成最佳。     

Advanced bacterial polyhydroxyalkanoates: Towards a versatile and sustainable platform for unnatural tailor-made polyesters

Polyhydroxyalkanoates (PHAs) are biopolyesters that generally consist of 3-, 4-, 5-, and 6-hydroxycarboxylic acids, which are accumulated as carbon and energy storage materials in many bacteria in limited growth conditions with excess carbon sources. Due to the diverse substrate specificities of PHA synthases, the key enzymes for PHA biosynthesis, PHAs with different material properties have been synthesized by incorporating different monomer components with differing compositions. Also, engineering PHA synthases using in vitro-directed evolution and site-directed mutagenesis facilitates the synthesis of PHA copolymers with novel material properties by broadening the spectrum of monomers available for PHA biosynthesis. Based on the understanding of metabolism of PHA biosynthesis, recombinant bacteria have been engineered to produce different types of PHAs by expressing heterologous PHA biosynthesis genes, and by creating and enhancing the metabolic pathways to efficiently generate precursors for PHA monomers. Recently, the PHA biosynthesis system has been expanded to produce unnatural biopolyesters containing 2-hydroxyacid monomers such as glycolate, lactate, and 2-hydroxybutyrate by employing natural and engineered PHA synthases. Using this system, polylactic acid (PLA), one of the major commercially-available bioplastics, can be synthesized from renewable resources by direct fermentation of recombinant bacteria. In this review, we discuss recent advances in the development of the PHA biosynthesis system as a platform for tailor-made polyesters with novel material properties.