Physiological and biochemical characteristics and capacity for polyhydroxyalkanoates synthesis in a glucose-utilizing strain of hydrogen-oxidizing bacteria, Ralstonia eutropha B8562

The physiological, biochemical, genetic, and cultural characteristics of the glucose-utilizing mutant strain Ralstonia eutropha B8562 were investigated in comparison with the parent strain R. eutropha B5786. The morphological, cultural, and biochemical characteristics of strain R. eutropha B8562 were similar to those of strain R. eutropha B5786. Genetic analysis revealed differences between the 16S rRNA gene sequences of these strains. The growth characteristics of the mutant using glucose as the sole carbon and energy source were comparable with those of the parent strain grown on fructose. Strain B8562 was characterized by high yields of polyhydroxyalkanoate (PHA) from different carbon sources (CO2, fructose, and glucose). In batch culture with glucose under nitrogen limitation, PHA accumulation reached 90% of dry weight. In PHA, beta-hydroxybutyrate was predominant (over 99 mol %); beta-hydroxyvalerate (0.25-0.72 mol %) and beta-hydroxyhexanoate (0.008-1.5 mol %) were present as minor components. The strain has prospects as a PHA producer on glucose-containing media.     

Physiological–biochemical properties and the ability to synthesize polyhydroxyalkanoates of the glucose-utilizing strain of the hydrogen bacterium Ralstonia eutropha B8562

Physiological–biochemical, genetic, and cultural properties of the glucose-utilizing mutant strain Ralstonia eutropha B8562 have been compared with those of its parent strain R. eutropha B5786. It has been shown that growth characteristics of the strain cultured on glucose as the sole carbon and energy source are comparable with those of the parent strain. Strain B8562 is characterized by high polyhydroxyalkanoate (PHA) yields on different carbon sources (CO₂, fructose, and glucose). PHA accumulation in the strain batch cultured on glucose under nitrogen deficiency reaches 90 %. The major monomer in the PHA is β-hydroxybutyric acid (more than 99 mol %); the identified minor components are β-hydroxyvaleric acid (0.25–0.72 mol %) and β-hydroxyhexanoic acid (0.08–1.5 mol %). The strain is a promising PHA producer on available sugar-containing media with glucose.     

Cloning and molecular organization of the polyhydroxyalkanoic acid synthase gene (phaC) of Ralstonia eutropha strain B5786

Class I polyhydroxyalkanoic acid (PHA) synthase gene (phaC) of Ralstonia eutropha strain B5786 was cloned and characterized. R. eutropha B5786 features the ability to synthesize multicomponent PHAs with short- and medium-chain-length monomers from simple carbohydrate substrate. A correlation was made between the molecular structure of PHA synthase and substrate specificity and the ability of strain-producers to accumulate PHAs of this or that structure. A strong similarity of PHA synthase of R. eutropha strain B5786 with PHA synthase of R. eutropha strain H16, which, as opposed to strain B5786, enables to incorporate medium chain length PHAs if hexanoate is used as carbon source, exhibited 99%. A correlation between the structure of PHA synthase of B5786 strain with synthases of microorganisms which synthesize short and medium chain length PHAs similarly to B5786 strain, showed an identity level from 26 to 41% (homology with synthase of Rhodospirillum rubrum makes 41%, Ectothiorhodospira shaposhnikovii makes 26%, Aeromonas punctata makes 40%, Thiococcus pfennigii makes 28%, Rhodococcus ruber makes 38%, and with PhaCl and PhaC2 synthases of Pseudomonas sp. 61–3 makes 34 and 37%, respectively). This allows for speaking about the absence of a direct connection between the molecular organization of PHA synthases and their functional abilities, namely, the ability to synthesize PHAs of a particular composition.     

Metabolite profiles of polyhydroxyalkanoate-producing Ralstonia eutropha H16

This study describes metabolite profiles of Ralstonia eutropha H16 focusing on biosynthesis of polyhydroxyalkanoates (PHAs), bacterial polyesters attracted as biodegradable bio-based plastics. As CoA-thioesters are important intermediates in PHA biosynthesis, four kinds of acyl-CoAs with medium chain length were prepared and used to establish analytical conditions for capillary electrophoresis-electron spray ionization-tandem mass spectrometry (CE–ESI-MS/MS). Metabolites were extracted from R. eutropha cells in growth, PHA production, and stationary phases on fructose and PHA production phase on octanoate, and subjected to stable isotope dilution-based comparative quantification by multiple reaction monitoring using CE–ESI-MS/MS and ¹³C-labeled metabolites prepared by extraction from R. eutropha mutant grown on U-¹³C₆-glucose. This procedure allowed to quantify relative changes of 94 ionic metabolites including CoA-thioesters. Hexose-phosphates except for glucose 1-phosphate were decreased in the PHA production phase than in the growth phase, suggesting reduced flux of sugar degradation after the cell growth. Several intermediates in TCA cycle and gluconeogenesis were increased in the PHA production phase on octanoate. Interestingly, ribulose 1,5-bisphosphate were detected in all the samples examined, raising possibilities of CO₂ fixation by Calvin–Benson–Bassham cycle in this bacterium even under heterotrophic growth conditions. Turnover of acyl moieties through β-oxidation was suggested to be active on fructose, as CoA-thioesters of C₆ and C₈ were detected in the fructose-grown cells. In addition, major metabolic pools in R. eutropha cells were estimated from the signal intensities. The results of the present study provided new insights into global metabolisms in PHA-producing R. eutropha.     

Biosynthesis of multicomponent polyhydroxyalkanoates by <Emphasis Type="Italic">Wautersia eutropha</Emphasis>

The effect of carbon supply on polyhydroxyalkanoate (PHA) synthesis by bacteria Wautersia eutropha was studied. Synthesis of multicomponent PHA composed of short-and long-chain monomers (C₄–C₈) by two natural strains (H16 and B5786) under mixotrophic conditions (CO₂ + alkanoic acids as cosubstrates) was demonstrated for the first time. The PHA composition was shown to be dependent on the cosubstrate type. In the presence of odd fatty acids, four-and five-component polymers were synthesized; hydroxybutyrate, hydroxyvalerate, and hydroxyheptanoate were the major monomers, while hydroxyhexanoate and hydroxyoctanoate were minor. In the presence of even fatty acids, PHA contained not only the corresponding molecules (hydroxyhexanoate and hydroxyoctanoate), but also hydroxyvalerate; synthesis of four-component PHA which contain mainly hydroxybutyrate and hydroxyhexanoate (up to 18 mol %) is therefore possible. A series of four-and five-component PHA was synthesized and their physicochemical characteristics were determined.     

Metabolic Engineering of Escherichia coli for Production of Enantiomerically Pure (R)-(−)-Hydroxycarboxylic Acids

A heterologous metabolism of polyhydroxyalkanoate (PHA) biosynthesis and degradation was established in Escherichia coli by introducing the Ralstonia eutropha PHA biosynthesis operon along with the R. eutropha intracellular PHA depolymerase gene. By with this metabolically engineered E. coli, enantiomerically pure (R)-3-hydroxybutyric acid (R3HB) could be efficiently produced from glucose. By employing a two-plasmid system, developed as the PHA biosynthesis operon on a medium-copy-number plasmid and the PHA depolymerase gene on a high-copy-number plasmid, R3HB could be produced with a yield of 49.5% (85.6% of the maximum theoretical yield) from glucose. By integration of the PHA biosynthesis genes into the chromosome of E. coli and by introducing a plasmid containing the PHA depolymerase gene, R3HB could be produced without plasmid instability in the absence of antibiotics. This strategy can be used for the production of various enantiomerically pure (R)-hydroxycarboxylic acids from renewable resources.     

Study of a Ralstonia eutropha Culture Producing Polyhydroxyalkanoates on Products of Coal Processing

Kinetic indices of growth, polyhydroxyalkanoate (PHA) accumulation, and gas exchange were studied in a culture of the carbon monoxide-resistant hydrogen strain Ralstonia eutropha B-5786 grown on a gaseous substrate (GS) obtained by lignite gasification. The GS was shown to be suitable for PHA production. To increase the degree of GS consumption, various modes of gas supply to the culture were tested. Based on the results, an algorithm was developed for calculating and controlling gas-exchange parameters in the PHA-accumulating culture of Ralstonia eutropha, grown on a new GS allowing high polymer yields (up to 75%) and degrees of substrate utilization (up to 90%).     

Genome-Based Analysis and Gene Dosage Studies Provide New Insight into 3-Hydroxy-4-Methylvalerate Biosynthesis in Ralstonia eutropha

Recombinant Ralstonia eutropha strain PHB⁻4 expressing the broad-substrate-specificity polyhydroxyalkanoate (PHA) synthase 1 from Pseudomonas sp. strain 61-3 (PhaC1_Ps) synthesizes a PHA copolymer containing the branched side-chain unit 3-hydroxy-4-methylvalerate (3H4MV), which has a carbon backbone identical to that of leucine. Mutant strain 1F2 was derived from R. eutropha strain PHB⁻4 by chemical mutagenesis and shows higher levels of 3H4MV production than does the parent strain. In this study, to understand the mechanisms underlying the enhanced production of 3H4MV, whole-genome sequencing of strain 1F2 was performed, and the draft genome sequence was compared to that of parent strain PHB⁻4. This analysis uncovered four point mutations in the 1F2 genome. One point mutation was found in the ilvH gene at amino acid position 36 (A36T) of IlvH. ilvH encodes a subunit protein that regulates acetohydroxy acid synthase III (AHAS III). AHAS catalyzes the conversion of pyruvate to 2-acetolactate, which is the first reaction in the biosynthesis of branched amino acids such as leucine and valine. Thus, the A36T IlvH mutation may show AHAS tolerance to feedback inhibition by branched amino acids, thereby increasing carbon flux toward branched amino acid and 3H4MV biosynthesis. Furthermore, a gene dosage study and an isotope tracer study were conducted to investigate the 3H4MV biosynthesis pathway. Based on the observations in these studies, we propose a 3H4MV biosynthesis pathway in R. eutropha that involves a condensation reaction between isobutyryl coenzyme A (isobutyryl-CoA) and acetyl-CoA to form the 3H4MV carbon backbone.     

Lipid and fatty acid metabolism in Ralstonia eutropha: relevance for the biotechnological production of value-added products

Lipid and fatty acid metabolism has been well studied in model microbial organisms like Escherichia coli and Bacillus subtilis. The major precursor of fatty acid biosynthesis is also the major product of fatty acid degradation (β-oxidation), acetyl-CoA, which is a key metabolite for all organisms. Controlling carbon flux to fatty acid biosynthesis and from β-oxidation allows for the biosynthesis of natural products of biotechnological importance. Ralstonia eutropha can utilize acetyl-CoA from fatty acid metabolism to produce intracellular polyhydroxyalkanoate (PHA). R. eutropha can also be engineered to utilize fatty acid metabolism intermediates to produce different PHA precursors. Metabolism of lipids and fatty acids can be rerouted to convert carbon into other value-added compounds like biofuels. This review discusses the lipid and fatty acid metabolic pathways in R. eutropha and how they can be used to construct reagents for the biosynthesis of products of industrial importance. Specifically, how the use of lipids or fatty acids as the sole carbon source in R. eutropha cultures adds value to these biotechnological products will be discussed here.     

Production of P(3-hydroxybutyrate-co-3-hydroxyhexanoate-co-3-hydroxyoctanoate) Terpolymers Using a Chimeric PHA Synthase in Recombinant Ralstonia eutropha and Pseudomonas putida

Recombinant strains of Ralstonia eutropha and Pseudomonas putida harboring a chimeric polyhydroxyalkanoate (PHA) synthase, which consisted of PHA synthases of Aeromonas caviae and R. eutropha, produced 3-hydroxybutyrate (3HB)-based PHA copolymers comprised of 3-hydroxyhexanoate and 3-hydroxyoctanoate units from dodecanoate (87–97 mol % 3HB), indicating that the chimeric PHA synthase possesses desirable substrate specificity leading to the production of 3HB-rich copolymers.     

Physicochemical properties of multicomponent poly(hydroxyalkanoates)

The properties of new five-component poly(hydroxyalkanoates) (PHA) formed by short-and medium-chain monomers synthesized by the bacterium Wautersia eutropha B5786 were studied by X-ray diffraction, IR spectroscopy, differential thermal analysis, and viscometry. The degree of crystallinity of PHA decreased from 72 to 57% as the molar fraction of hydroxyhexanoate increased from 2.5 to 18.0 mol%. The melting temperature (T _m) and decomposition temperature (T _d) of the multicomponent PHA are lower than those for poly(hydroxybutyrate), whose T _m and T _d are 168–170 and 260–265°C, respectively. Both parameters of the multicomponent PHA decrease to 156 and 252°C, respectively, as the hydroxyhexanoate mole fraction is raised. The effect of hydroxyhexanoate on the physicochemical properties of the PHA is similar to that of hydroxyvalerate observed previously.     

In-silico-driven metabolic engineering of Pseudomonas putida for enhanced production of poly-hydroxyalkanoates

Here, we present systems metabolic engineering driven by in-silico modeling to tailor Pseudomonas putida for synthesis of medium chain length PHAs on glucose. Using physiological properties of the parent wild type as constraints, elementary flux mode analysis of a large-scale model of the metabolism of P. putida was used to predict genetic targets for strain engineering. Among a set of priority ranked targets, glucose dehydrogenase (encoded by gcd) was predicted as most promising deletion target. The mutant P. putida Δgcd, generated on basis of the computational design, exhibited 100% increased PHA accumulation as compared to the parent wild type, maintained a high specific growth rate and exhibited an almost unaffected gene expression profile, which excluded detrimental side effects of the modification. A second mutant strain, P. putida Δpgl, that lacked 6-phosphogluconolactonase, exhibited a substantially decreased PHA synthesis, as was also predicted by the model. The production potential of P. putida Δgcd was assessed in batch bioreactors. The novel strain showed an increase of the PHA yield (+80%), the PHA titer (+100%) and cellular PHA content (+50%) and revealed almost unaffected growth and diminished by-product formation. It was thus found superior in all relevant criteria towards industrial production. Beyond the contribution to more efficient PHA production processes at reduced costs that might replace petrochemical plastics in the future, the study illustrates the power of computational prediction to tailor microbial strains for enhanced biosynthesis of added-value compounds.     

Roles of Multiple Acetoacetyl Coenzyme A Reductases in Polyhydroxybutyrate Biosynthesis in Ralstonia eutropha H16

The bacterium Ralstonia eutropha H16 synthesizes polyhydroxybutyrate (PHB) from acetyl coenzyme A (acetyl-CoA) through reactions catalyzed by a β-ketothiolase (PhaA), an acetoacetyl-CoA reductase (PhaB), and a polyhydroxyalkanoate synthase (PhaC). An operon of three genes encoding these enzymatic steps was discovered in R. eutropha and has been well studied. Sequencing and analysis of the R. eutropha genome revealed putative isologs for each of the PHB biosynthetic genes, many of which had never been characterized. In addition to the previously identified phaB1 gene, the genome contains the isologs phaB2 and phaB3 as well as 15 other potential acetoacetyl-CoA reductases. We have investigated the roles of the three phaB isologs by deleting them from the genome individually and in combination. It was discovered that the gene products of both phaB1 and phaB3 contribute to PHB biosynthesis in fructose minimal medium but that in plant oil minimal medium and rich medium, phaB3 seems to be unexpressed. This raises interesting questions concerning the regulation of phaB3 expression. Deletion of the gene phaB2 did not result in an observable phenotype under the conditions tested, although this gene does encode an active reductase. Addition of the individual reductase genes to the genome of the ΔphaB1 ΔphaB2 ΔphaB3 strain restored PHB production, and in the course of our complementation experiments, we serendipitously created a PHB-hyperproducing mutant. Measurement of the PhaB and PhaA activities of the mutant strains indicated that the thiolase reaction is the limiting step in PHB biosynthesis in R. eutropha H16 during nitrogen-limited growth on fructose.Polyhydroxyalkanoates (PHAs) are natural polyesters synthesized by a wide range of bacteria as carbon and energy reserves. PHAs are typically stored when organisms are in an environment in which carbon is plentiful but the lack of another nutrient limits normal cell growth. It has been found that in environments with fluctuating carbon levels, PHA producers have crucial advantages over rival species (14). In addition to their importance in the microbial world, these polymers have been studied for their potential uses in biodegradable consumer goods (12) and medical products (22) and as chemical precursors (4). Although many PHA monomers have been discovered, the most common are 3-hydroxyalkanoates (32). Common PHAs are typically characterized by their constituent monomers as short-chain-length polymers (SCL-PHA; C₄ and C₅ monomers) or medium-chain-length polymers (MCL-PHA; C₆ and longer monomers).The model organism used to study PHA biosynthesis is the Gram-negative bacterium Ralstonia eutropha. This organism accumulates a high percentage of its cell dry weight (CDW) as SCL-PHA under nutrient limitation. When grown on sugars or plant oils, R. eutropha makes poly(3-hydroxybutyrate) (PHB) almost exclusively, although the addition of precursors such as propionate to the growth medium can lead to incorporation of 3-hydroxyvalerate into the polymer chain as well (2). An operon of biosynthetic genes from R. eutropha encoding enzymes sufficient for synthesis of PHB from acetyl coenzyme A (acetyl-CoA), which consisted of phaC-phaA-phaB, was discovered in the late 1980s (25, 26, 36). In this pathway, two molecules of acetyl-CoA are condensed by a β-ketothiolase (PhaA) and the resulting acetoacetyl-CoA is reduced by a reductase (PhaB) to form (R)-3-hydroxybutyryl-CoA (HB-CoA), which is the substrate for the PHA synthase (PhaC). Sequencing and analysis of the R. eutropha genome revealed the existence of putative isologs for each of the PHA synthetic genes (29). While the existence of alternate β-ketothiolases was already known (39), most of the potential isologs identified had never been characterized.Our group wanted to better understand how acetoacetyl-CoA reduction occurs in R. eutropha. In addition to the earlier-identified phaB gene, now referred to as phaB1 (GeneID, 4249784), the genes phaB2 (GeneID, 4249785) and phaB3 (GeneID, 4250155) were discovered on R. eutropha chromosome 1. Fifteen other potential isologs were also found to encode amino acid sequences that could potentially indicate acetoacetyl-CoA reductase activity (29). The roles of the newly discovered genes in PHB biosynthesis were unclear, especially given the results of an earlier biochemical study that suggested there was a single NADPH-dependent acetoacetyl-CoA reductase in R. eutropha (10). In order to determine the roles of the reductase genes in R. eutropha, we deleted phaB1, phaB2, and phaB3 from the genome both individually and in combination. In addition to characterizing these newly discovered genes, we also hoped to eliminate or diminish formation of HB-CoA by stopping the reduction reaction. Efforts to purify the PHA synthase from R. eutropha have been complicated by the high levels of PHB made by this organism (7). Studying formation and growth of PHB granules is difficult because PHB accumulates at a high rate, causing individual granules to coalesce and become indistinct (44). We therefore believed that an R. eutropha strain with decreased HB-CoA synthesis would be a useful experimental tool and could also serve as a platform for engineering new PHA synthesis pathways into R. eutropha.     

Sequence of PHA synthase gene from two strains of Rhodospirillum rubrum and in vivo substrate specificity of four PHA synthases across two heterologous expression systems

A 3.0-kb genomic fragment has been isolated from Rhodospirillum rubrum (ATCC 25903) that contains an open reading frame (ORF) with strong homology to other known polyhydroxyalkanoate (PHA) synthase genes. This ORF has lower homology to the R. rubrum strain Ha PHA synthase than would be expected within the same species. We have conducted a series of heterologous expression studies evaluating the in vivo substrate specificity of PHA synthase genes from Rhodobacter sphaeroides, Ralstonia eutropha (formerly Alcaligenes eutrophus), Thiocystis violacea, and Nocardia corrallina, within the PHA-synthase-negative hosts, Ralstonia eutropha DSM541 and Pseudomonas putida GpP104. The N. corrallina PHA synthase incorporated the highest percentage of C5 monomers in the polymer when fermented in medium supplemented with 0.1% heptanoate as the sole carbon source. When the T. violacea and R. sphaeroides were expressed in the PHA-negative host DSM541, a greater percentage of C5 monomer was observed in the polymer as compared to the expression of the PHA synthase of R. eutropha, when the transconjugants were fermented in medium supplemented with 0.4% propionate. Evaluation for preference of medium-chain-length monomers demonstrated the flexibility of the N. corrallina, T. violacea, and R. eutropha synthase genes to polymerize a copolyester composed of short- and medium-chain-length monomers when the respective transconjugants were fermented in medium supplemented with 0.5% octanoate. These studies demonstrate that the PHA synthase from N. corrallina, T. violacea, and R. eutropha are able to polymerize a copolyester composed of short- and medium-chain-length monomers, while the PHA synthase from R. sphaeroides lacks this ability and only produces a short-chain-length polymer. These observations suggest that the composition of the PHA from the PHA-producing organisms does not necessarily reflect the inherent specificity of the PHA synthase. Received: 16 March 1999 / Received revision: 24 August 1999 / Accepted: 24 September 1999     

Metabolic engineering of Escherichia coli for biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from glucose

The Escherichia coli XL1-blue strain was metabolically engineered to synthesize poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] through 2-ketobutyrate, which is generated via citramalate pathway, as a precursor for propionyl-CoA. Two different metabolic pathways were examined for the synthesis of propionyl-CoA from 2-ketobutyrate. The first pathway is composed of the Dickeya dadantii 3937 2-ketobutyrate oxidase or the E. coli pyruvate oxidase mutant (PoxB L253F V380A) for the conversion of 2-ketobutyrate into propionate and the Ralstonia eutropha propionyl-CoA synthetase (PrpE) or the E. coli acetyl-CoA:acetoacetyl-CoA transferase for further conversion of propionate into propionyl-CoA. The second pathway employs pyruvate formate lyase encoded by the E. coli tdcE gene or the Clostridium difficile pflB gene for the direct conversion of 2-ketobutyrate into propionyl-CoA. As the direct conversion of 2-ketobutyrate into propionyl-CoA could not support the efficient production of P(3HB-co-3HV) from glucose, the first metabolic pathway was further examined. When the recombinant E. coli XL1-blue strain equipped with citramalate pathway expressing the E. coli poxB L253F V380A gene and R. eutropha prpE gene together with the R. eutropha PHA biosynthesis genes was cultured in a chemically defined medium containing 20 g/L of glucose as a sole carbon source, P(3HB-co-2.3 mol% 3HV) was produced up to the polymer content of 61.7 wt.%. Moreover, the 3HV monomer fraction in P(3HB-co-3HV) could be increased up to 5.5 mol% by additional deletion of the prpC and scpC genes, which are responsible for the metabolism of propionyl-CoA in host strains.     

Establishment of an alternative phosphoketolase-dependent pathway for fructose catabolism in Ralstonia eutropha H16

The β-proteobacterium Ralstonia eutropha H16 utilizes fructose and gluconate as carbon sources for heterotrophic growth exclusively via the Entner–Doudoroff pathway with its key enzyme 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase. By deletion of the responsible gene eda, we constructed a KDPG aldolase-negative strain, which is disabled to supply pyruvate for energy metabolism from fructose or gluconate as sole carbon sources. To restore growth on fructose, an alternative pathway, similar to the fructose-6-phosphate shunt of heterofermentative bifidobacteria, was established. For this, the xfp gene from Bifidobacterium animalis, coding for a bifunctional xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp; Meile et al. in J Bacteriol 183:2929–2936, 2001), was expressed in R. eutropha H16 PHB⁻4 Δeda. This Xfp catalyzes the phosphorolytic cleavage of fructose 6-phosphate to erythrose 4-phosphate and acetylphosphate as well as of xylulose 5-phosphate to glyceralaldehyde 3-phosphate and acetylphosphate. The recombinant strain showed phosphoketolase (PKT) activity on either substrate, and was able to use fructose as sole carbon source for growth, because PKT is the only enzyme that is missing in R. eutropha H16 to establish the artificial fructose-6-phosphate shunt. The Xfp-expressing strain R. eutropha H16 PHB⁻4 Δeda (pBBR1MCS-3::xfp) should be applicable for a novel variant of a plasmid addiction system to stably maintain episomally encoded genetic information during fermentative production processes. Plasmid addiction systems are often used to ensure plasmid stability in many biotechnology relevant microorganisms and processes without the need to apply external selection pressure, like the addition of antibiotics. By episomal expression of xfp in a R. eutropha H16 mutant lacking KDPG aldolase activity and cultivation in mineral salt medium with fructose as sole carbon source, the growth of this bacterium was addicted to the constructed xfp harboring plasmid. This novel selection principle extends the applicability of R. eutropha H16 as production platform in biotechnological processes.     

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.     

Ethanol Production by Thermophilic Bacteria: Physiological Comparison of Solvent Effects on Parent and Alcohol-Tolerant Strains of Clostridium thermohydrosulfuricum

The effects of temperature, solvents, and cultural conditions on the fermentative physiology of an ethanol-tolerant (56 g/liter at 60°C) and parent strain of Clostridium thermohydrosulfuricum were compared. An ethanol-tolerant mutant was selected by successive transfer of the parent strain into media with progressively higher ethanol concentrations. Physiological differences noted in the mutant included enhanced growth, tolerance to various solvents, and alterations in the substrate range and the fermentation end product ratio. Ethanol tolerance was temperature dependent in the mutant but not in the parent strain. The mutant grew with ethanol concentrations up to 8.0% (wt/vol) at 45°C, but only up to 3.3% (wt/vol) at 68°C. Low ethanol concentration (0.2 to 1.6% [wt/vol]) progressively inhibited the parent strain to where glucose was not fermented at 2.0% (wt/vol) ethanol. Both strains grew and produced alcohols on glucose complex medium at 60°C in the presence of either 5% methanol or acetone, and these solvents when added at low concentration stimulated fermentative metabolism. The mutant produced ethanol at high concentrations and displayed an ethanol/glucose ratio (mole/mole) of 1.0 in media where initial ethanol concentrations were ≤4.0% (wt/vol), whereas when ethanol concentration was changed from 0.1% to 1.6% (wt/vol), the ethanol/glucose ratio for the parent strain changed from 1.6 to 0.6. These data indicate that C. thermohydrosulfuricum strains are tolerant of solvents and that low ethanol tolerance is not a result of disruption of membrane fluidity or glycolytic enzyme activity.     

Biosynthesis and Characterization of Poly(3-hydroxybutyrate-co-3- hydroxyhexanoate) from Palm Oil Products in a Wautersia eutropha Mutant

Palm kernel oil, palm olein, crude palm oil and palm acid oil were used for the synthesis of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] by a mutant strain of Wautersia eutropha (formerly Ralstonia eutropha) harboring the Aeromonas caviae polyhydroxyalkanoate (PHA) synthase gene. Palm kernel oil was an excellent carbon source for the production of cell biomass and P(3HB-co-3HHx). About 87% (w/w) of the cell dry weight as P(3HB-co-3HHx) was obtained using 5 g palm kernel oil/l. Gravimetric and microscopic analyses further confirmed the high PHA content in the recombinant cells. The molar fraction of 3HHx remained constant at 5 mol % regardless of the type and concentration of palm oil products used. The small amount of 3HHx units was confirmed by ¹³C NMR analysis. The number average molecular weight (M_n) of the PHA copolymer produced from the various palm oil products ranged from 27 0000 to 46 0000 Da. The polydispersity was in the range of 2.6–3.9.