Physical properties of microbial polythioesters: characterization of poly(3-mercaptoalkanoates) synthesized by engineered Escherichia coli

Physical properties of chiral poly(thioesters), PTEs, prepared by engineered Escherichia coli, were examined by GPC, 13C CP/MAS solid-state NMR, X-ray diffraction, and thermal analysis. Microbial homopolymers of PTEs, poly(3-mercaptopropionate), PMP, and poly(3-mercaptovalerate), PMV, showed different solubility characteristics compared to poly(hydroxyalkanoates), PHAs. Generally, PTEs required higher temperatures for dissolution. Poly(3-mercaptobutyrate), PMB, and PMV dissolve in chloroform, and the molecular weight values were revealed by GPC as 176,000 and 165,000, respectively. The density values for PMP and PMB were 1.42 and 1.27 g/cm3, respectively. These values are similar to those for oxygen analogues. The NMR spectra for PTEs showed that carbonyl carbons are greatly shifted downfield by the sulfur atoms in the chain backbone compared to the PHA family. X-ray powder diffraction data indicated that PTEs are crystalline materials, but they do not crystallize as well as in the PHA family. The melting point, Tm, for PMP was 170 degrees C, which is about 100 degrees C higher than the equivalent oxygen analogue, poly(3-hydroxypropionate), PHP, and almost the same as that of bacterial poly(3-hydroxybutyrate), PHB. According to thermal analysis, only the PMP sample had enhanced heat stability, e.g., the decomposition temperature for PMP was 277 degrees C at 5% weight loss, whereas the values for PHP and PHB were 233 and 260 degrees C at the same weight loss, respectively.     

The synthesis of hydroxybutyrate and hydroxyvalerate copolymers by the bacterium Ralstonia eutropha

The paper deals with the study of the synthesis of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) copolymers by the bacterium Ralstonia eutropha B-5786 grown under different carbon nutrition conditions (growth on carbon dioxide, fructose, and CO2-valerate and fructose-valerate mixtures). The parameters to be analyzed included the yield of biomass, the yield, synthesis rate, and composition of copolymers, the activity of the key enzymes of polyhydroxyalkanoate (PHA) synthesis (beta-ketothiolase, acetoacetyl-CoA reductase, and PHA synthase), the maximum tolerable concentration of valerate to the bacterium, and the conditions that govern the incorporation of hydroxyvalerate to copolymers. This allowed the relationship between cultivation conditions and the proportion of monomers in the copolymers to be deduced. We were able to synthesize a range of 3HB/3HV copolymers and found that the thermal characteristics and the degree of crystallinity of these copolymers depend on the molar fraction of 3HV.     

The synthesis of hydroxybutyrate and hydroxyvalerate copolymers by the bacterium Ralstonia eutropha

The paper deals with the study of the synthesis of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) copolymers by the bacterium Ralstonia eutropha B-5786 grown under different carbon nutrition conditions (growth on carbon dioxide, fructose, and CO₂-valerate and fructose-valerate mixtures). The parameters to be analyzed included the yield of biomass; the yield, synthesis rate, and composition of copolymers; the activity of the key enzymes of polyhydroxyalkanoate (PHA) synthesis -ketothiolase, acetoacetyl-CoA reductase, and PHA synthase); the maximum tolerable concentration of valerate to the bacterium; and the conditions that govern the incorporation of hydroxyvalerate into copolymers. This allowed the relationship between cultivation conditions and the proportion of monomers in the copolymers to be deduced. We were able to synthesize a range of 3HB/3HV copolymers and found that the thermal characteristics and the degree of crystallinity of such copolymers depend on the molar fraction of 3HV.Translated from Mikrobiologiya, Vol. 74, No. 1, 2005, pp. 63–69.Original Russian Text Copyright © 2005 by Volova, Kalacheva.     

Ethanethiol degradation by Ralstonia eutropha

In the present study, a pure culture of Ralstonia eutropha was used to degrade gaseous ethanethiol. Ethane thiol at various initial concentrations ranging from 115 to 320 mg/m³ was degraded almost completely within 120 ～ 168 h, while at higher concentrations up to 452 mg/m³, removal efficiency declined. It was likely that ethanethiol was used as the source of energy by R. eutropha, since no clear increase in the biomass concentration was observed. Kinetic data of ethanethiol bidegradation could be fitted using the Monod model. The kinetic parameters were q _m = 0.23 (mg ethanethiol/g biomass/h), and K _s = 1.379 (mg/L). The mineralization pathway of ethanethiol through sulphate, as the detected product, and the energy production were discussed in some detail.     

Novel precursor substrates for polythioesters (PTE) and limits of PTE biosynthesis in Ralstonia eutropha

A novel class of biopolymers referred to as polythioesters (PTE) was recently detected when the polyhydroxyalkanoate (PHA) accumulating bacterium Ralstonia eutropha was cultivated in the presence of 3-mercaptopropionic acid (3MP) or 3,3'-thiodipropionic acid (TDP). In this study, 3,3'-dithiodipropionic acid (DTDP) and 3-mercaptovaleric acid (3MV) were identified as two additional precursor carbon sources for in vivo biosynthesis of PTE in R. eutropha. Biosynthesis of copolymers of 3-hydroxybutyrate (3HB) and 3MP, which contributed 19-25% of cell dry matter, was compared referring to the different precursor substrates. Using DTDP as carbon source, which is probably cleaved into two molecules 3MP, yielded an about 2.3-fold higher molar 3MP content of the copolyester than TDP, which is probably cleaved into only one molecule 3MP. Furthermore, cultivation of R. eutropha in the presence of 3MV resulted in biosynthesis of copolymers consisting predominantly of 3HB with low amounts of 3MV and 3-hydroxyvalerate, each contributing less than 5 mol% of the constituents. In contrast, 4-mercaptobutyric acid could be not incorporated into PHAs, although - as documented in this study - five different strategies, various precursor substrates, R. eutropha and also a recombinant strain of Escherichia coli were employed. Therefore, this study not only extended the range of substrates suitable for PTE biosynthesis and also the range of PTE constituents in R. eutropha, it also demonstrates limits for PTE biosynthesis in this bacterium.     

Characterization of propionate CoA-transferase from Ralstonia eutropha H16

In this study, a propionate CoA-transferase (H16_A2718; EC 2.8.3.1) from Ralstonia eutropha H16 (Pct _Re ) was characterized in detail. Glu342 was identified as catalytically active amino acid residue via site-directed mutagenesis. Activity of Pct _Re was irreversibly lost after the treatment with NaBH₄ in the presence of acetyl-CoA as it is shown for all CoA-transferases from class I, thereby confirming the formation of the covalent enzyme-CoA intermediate by Pct _Re . In addition to already known CoA acceptors for Pct _Re such as 3-hydroxypropionate, 3-hydroxybutyrate, acrylate, succinate, lactate, butyrate, crotonate and 4-hydroxybutyrate, it was found that glycolate, chloropropionate, acetoacetate, valerate, trans-2,3-pentenoate, isovalerate, hexanoate, octanoate and trans-2,3-octenoate formed also corresponding CoA-thioesters after incubation with acetyl-CoA and Pct _Re . Isobutyrate was found to be preferentially used as CoA acceptor amongst other carboxylates tested in this study. In contrast, no products were detected with acetyl-CoA and formiate, bromopropionate, glycine, pyruvate, 2-hydroxybutyrate, malonate, fumarate, itaconate, β-alanine, γ-aminobutyrate, levulate, glutarate or adipate as potential CoA acceptor. Amongst CoA donors, butyryl-CoA, crotonyl-CoA, 3-hydroxybutyryl-CoA, isobutyryl-CoA, succinyl-CoA and valeryl-CoA apart from already known propionyl-CoA and acetyl-CoA could also donate CoA to acetate. The highest rate of the reaction was observed with 3-hydroxybutyryl-CoA (2.5 μmol mg^?1 min^?1). K _m values for propionyl-CoA, acetyl-CoA, acetate and 3-hydroxybutyrate were 0.3, 0.6, 4.5 and 4.3 mM, respectively. The rather broad substrate range might be a good starting point for enzyme engineering approaches and for the application of Pct _Re in biotechnological polyester production.     

A novel high-cell-density protein expression system based on Ralstonia eutropha

We describe the development of a novel protein expression system based on the industrial fermentation organism Ralstonia eutropha (formerly known as Alcaligenes eutrophus) NCIMB 40124. This new system overcomes some of the shortcomings of traditional Escherichia coli-based protein expression systems, particularly the propensity of such systems to form inclusion bodies during high-level expression. Using a proteomics approach, we identified promoters that can be induced by simple process parameters or medium compositions in high-density cell culture or shake flasks, respectively. By combining newly developed molecular biological tools with a high-cell-density fermentation process, we were able to produce high levels (>1 g/liter) of soluble, active organophosphohydrolase, a model enzyme prone to inclusion body formation in E. coli.     

Catabolism of 3-Nitrophenol by Ralstonia eutropha JMP 134

Ralstonia eutropha JMP 134 utilizes 3-nitrophenol as the sole source of nitrogen, carbon, and energy. The entire catabolic pathway of 3-nitrophenol is chromosomally encoded. An initial NADPH-dependent reduction of 3-nitrophenol was found in cell extracts of strain JMP 134. By use of a partially purified 3-nitrophenol nitroreductase from 3-nitrophenol-grown cells, 3-hydroxylaminophenol was identified as the initial reduction product. Resting cells of R. eutropha JMP 134 metabolized 3-nitrophenol to N-acetylaminohydroquinone under anaerobic conditions. With cell extracts, 3-hydroxylaminophenol was converted into aminohydroquinone. This enzyme-mediated transformation corresponds to the acid-catalyzed Bamberger rearrangement. Enzymatic conversion of the analogous hydroxylaminobenzene yields a mixture of 2- and 4-aminophenol.     

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.     

Production of polyhydroxybutyrate by Ralstonia eutropha from protein hydrolysates

Polyhydroxybutyrate (PHB) was produced by Ralstonia eutropha DSM 11348 (formerly Alicaligenes eutrophus) in media containing 20–30 g l⁻¹ casein peptone or casamino acids as sole sources of nitrogen. In fermentations using media based on casein peptone, permanent growth up to a cell dry mass of 65 g l⁻¹ was observed. PHB accumulated in cells up to 60%–80% of dry weight. The lowest yields were found in media without any trace elements or with casamino acids added only. The residual cell dry masses were limited to 10–15 g l⁻¹ and did not contain PHB. The highest productivity amounted to 1.2 g PHB l⁻¹ h⁻¹. The mean molecular mass of the biopolymer was determined as 750 kDa. The proportion of polyhydroxyvalerate was less than 0.2% in PHB. The bioprocess was scaled up to a 300-l plant. During a fermentation time of 39 h the cells accumulated PHB to 78% w/w. The productivity was 0.98 g PHB l⁻¹ h¹. Received: 8 July 1998 / Accepted: 26 August 1998     

Characterization of an extracellular lipase and its chaperone from Ralstonia eutropha H16

Lipase enzymes catalyze the reversible hydrolysis of triacylglycerol to fatty acids and glycerol at the lipid–water interface. The metabolically versatile Ralstonia eutropha strain H16 is capable of utilizing various molecules containing long carbon chains such as plant oil, organic acids, or Tween as its sole carbon source for growth. Global gene expression analysis revealed an upregulation of two putative lipase genes during growth on trioleate. Through analysis of growth and activity using strains with gene deletions and complementations, the extracellular lipase (encoded by the lipA gene, locus tag H16_A1322) and lipase-specific chaperone (encoded by the lipB gene, locus tag H16_A1323) produced by R. eutropha H16 was identified. Increase in gene dosage of lipA not only resulted in an increase of the extracellular lipase activity, but also reduced the lag phase during growth on palm oil. LipA is a non-specific lipase that can completely hydrolyze triacylglycerol into its corresponding free fatty acids and glycerol. Although LipA is active over a temperature range from 10 °C to 70 °C, it exhibited optimal activity at 50 °C. While R. eutropha H16 prefers a growth pH of 6.8, its extracellular lipase LipA is most active between pH 7 and 8. Cofactors are not required for lipase activity; however, EDTA and EGTA inhibited LipA activity by 83 %. Metal ions Mg²⁺, Ca²⁺, and Mn²⁺ were found to stimulate LipA activity and relieve chelator inhibition. Certain detergents are found to improve solubility of the lipid substrate or increase lipase-lipid aggregation, as a result SDS and Triton X-100 were able to increase lipase activity by 20 % to 500 %. R. eutropha extracellular LipA activity can be hyper-increased, making the overexpression strain a potential candidate for commercial lipase production or in fermentations using plant oils as the sole carbon source.     

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。     

Degradation of formaldehyde in packed-bed bioreactor by kissiris-immobilized Ralstonia eutropha

The ability of the Ralstonia eutropha cells to utilize formaldehyde (FA) as the only source of carbon and energy was studied in the kissiris-immobilized cell bioreactor (KICB) in batch-recirculation and continuous modes of operation. In batch-recirculation experiments, the test bacterium could tolerate concentrations of FA up to 1,400 mg/L at 30°C and aeration rate equal to 0.75 vvm (r _S = 7.25 mg/L/h, q S = 0.019 g_FA/g_cell/h). However, further increase of initial FA concentration resulted in degradation reaction of FA to stop at 1,600 mg/L. Results of continuous mode experiments showed that the biodegradation performance of the KICB was dependent on both feed flow rate and inlet FA concentration parameters. The optimum feed flow rate which corresponded to the highest biodegradation rate (r _S = 240.3 mg/L/h) was observed at Q = 18 mL/min when KICB did not operate under the external mass transfer limiting regime. Substrate inhibition kinetics (Edwards and Luong equations) were used to describe the experimental specific degradation rates data. According to the Luong model, the values of the maximum specific degradation rate (q _max), half-saturation coefficient (K _S), the maximum allowable FA concentration (S _m), and the shape factor (n) were 0.178 g_FA/g_cell/h, 250.9 mg/L, 1,600 mg/L, and 1.86, respectively.     

Conversion of volatile fatty acids into polyhydroxyalkanoate by Ralstonia eutropha

Aims:  The aims of this study were to optimize condensed corn solubles (CCS) as a medium for growth of Ralstonia eutropha and to determine the effects of individual volatile fatty acids (VFAs) on polyhydroxyalkanoate (PHA) production .
Methods and Results:  A CCS medium of concentration 240 g l⁻¹ with a carbon : nitrogen ratio of 50 : 1 was developed as the optimal medium. Cultures were grown in 1-l aerated flasks at 250 rev min⁻¹ at 30°C for 120 h. Comparable growth rates were observed in CCS vs a defined medium. At 48 h, VFAs were fed individually at different levels. Optimal levels of all the acids were determined to maximize PHA production. An overall comparison of the VFAs indicated that butyric and propionic acids provided the best results.
Conclusion:  An optimized CCS medium supported growth of R. eutropha . Butyric and propionic acids were the most efficient carbon sources to maximize PHA production when added at the 5 g l⁻¹ level.
Significance and Impact of the Study:  The study shows that a byproduct of ethanol industry can be effectively used as a low cost medium for PHA production, thus partly reducing the cost of commercialization of biopolymers.     

Enzymatic recovery and purification of polyhydroxybutyrate produced by Ralstonia eutropha

Polyhydroxybutyrate (PHB) is the most studied among a wide variety of polyhydroxyalkanoates, bacterial biodegradable polymers known as potential substitutes for conventional plastics. This work aimed at evaluating the use of enzymes to recover and purify the PHB produced by Ralstonia eutropha DSM545. Screening experiments allowed the selection of trypsin, bromelain and lysozyme among six enzymes, based on their efficiency in lysing cells of a non-PHB producing R. eutropha strain. Then, process conditions for high efficiency in PHB purification from the DSM545 cells were searched for the enzymes previously selected. The best result was achieved with 2.0% of bromelain (enzyme mass per biomass), equivalent to 14.1 U ml(-1), at 50 degrees C and pH 9.0, resulting in 88.8% PHB purity. Aiming at improving the process efficiency and reducing the enzyme cost, experiments were carried out with pancreatin, leading to 90.0% polymer purity and an enzyme cost three times lower than the one obtained with bromelain. The molecular mass analysis of PHB showed no polymer degradation. Therefore, this work demonstrates the potential of using enzymes in order to recover and purify PHB and bacterial biopolymers in general.     

Biosynthesis of short-chain-length-polyhydroxyalkanoates during the dual-nutrient-limited zone by Ralstonia eutropha

Biosynthesis of PHAs by Raltonia eutropha during the dual nutrient-limitation-zone was investigated with mixed organic acids as carbon sources and (NH₄)₂SO₄ as nitrogen source. Two different methods of maintaining the dual-nutrient-limitation zone were adopted by feeding mixed acids and (NH₄)₂SO₄ at determined rates into the fermentation cultures which were initially free of carbon sources (method A) or nitrogen sources (method B). The results indicate that, firstly, with the increase of the width of the dual-nutrient-limitation zone, the yield of short-chain-length-polyhydroxyalkanoates also increases and it suggests that most of the short-chain-length-polyhydroxyalkanoates were biosynthesized during the dual-nutrient-limitation zone. Secondly, in contrast with the dual-nutrient-limitation method of limiting the nitrogen source first (method B), the dual-nutrient-limitation method of limiting the carbon source first (method A) was more favourable for the production of short-chain-length-polyhydroxyalkanoates, and the maximum production of short-chain-length-polyhydroxyalkanoates of these two methods are 3.72 and 2.55 g/l, respectively.     

Characterization and functional analyses of R-specific enoyl coenzyme A hydratases in polyhydroxyalkanoate-producing Ralstonia eutropha

A genome survey of polyhydroxyalkanoate (PHA)-producing Ralstonia eutropha H16 detected the presence of 16 orthologs of R-specific enoyl coenzyme A (enoyl-CoA) hydratase, among which three proteins shared high homologies with the enzyme specific to enoyl-CoAs of medium chain length encoded by phaJ4 from Pseudomonas aeruginosa (phaJ4(Pa)). The recombinant forms of the three proteins, termed PhaJ4a(Re) to PhaJ4c(Re), actually showed enoyl-CoA hydratase activity with R specificity, and the catalytic efficiencies were elevated as the substrate chain length increased from C(4) to C(8). PhaJ4a(Re) and PhaJ4b(Re) showed >10-fold-higher catalytic efficiency than PhaJ4c(Re). The functions of the new PhaJ4 proteins were investigated using previously engineered R. eutropha strains as host strains; these strains are capable of synthesizing poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) [P(3HB-co-3HHx)] from soybean oil. Deletion of phaJ4a(Re) from the chromosome resulted in significant decrease of 3HHx composition in the accumulated copolyester, whereas no change was observed with deletion of phaJ4b(Re) or phaJ4c(Re), indicating that only PhaJ4a(Re) was one of the major enzymes supplying the (R)-3HHx-CoA monomer through β-oxidation. Introduction of phaJ4a(Re) or phaJ4b(Re) into the R. eutropha strains using a broad-host-range vector enhanced the 3HHx composition of the copolyesters, but the introduction of phaJ4c(Re) did not. The two genes were then inserted into the pha operon on chromosome 1 of the engineered R. eutropha by homologous recombination. These modifications enabled the biosynthesis of P(3HB-co-3HHx) composed of a larger 3HHx fraction without a negative impact on cell growth and PHA production on soybean oil, especially when phaJ4a(Re) or phaJ4b(Re) was tandemly introduced with phaJ(Ac) from Aeromonas caviae.