Microbial Production of Poly-beta-Hydroxybutyric Acid from d-Xylose and Lactose by Pseudomonas cepacia

Poly-beta-hydroxybutyric acid (PHB) was produced from xylose and lactose by using Pseudomonas cepacia. Approximately 50% PHB (grams of PHB total/grams of biomass total) was produced. With a laser-based fluorescent probe, beta-galactosidase activity was shown to be induced in P. cepacia cells grown on lactose but not in those grown on glucose or xylose. P. cepacia has the potential to produce biodegradable thermoplastics from hemicellulosic hydrolysates and cheese whey.     

Production of Poly-β-Hydroxyalkanoic Acid by Pseudomonas cepacia

The possibility of using the nutritionally versatile bacterium Pseudomonas cepacia to produce poly-β-hydroxyalkanoic acid was evaluated. Chemostat culture showed that growth of P. cepacia became nitrogen limited when the molar carbon-to-nitrogen ratio of the medium fed into the fermentor was above 15. When grown under nitrogen limitation in batch culture with fructose as the sole source of carbon, P. cepacia accumulated poly-β-hydroxybutyric acid (PHB) in excess of 50% of the dry weight of its biomass. In batch culture, almost no PHB was produced until the onset of nitrogen limitation. After this point, PHB was produced at a linear rate of 0.12 g liter⁻¹ h⁻¹ (from a constant value of 1.6 g of cellular protein liter⁻¹). PHB produced by P. cepacia had a weight-average molecular weight of 5.37 × 10⁵ g mol⁻¹ and a polydispersivity index of 3.9. Poly(β-hydroxybutyric acid-β-hydroxyvaleric acid) copolymer was produced with a poly-β-hydroxybutyric acid-poly-β-hydroxyvaleric acid ratio of up to 30% by weight when propionic acid was added to the medium.     

Biosynthesis of Poly-β-Hydroxyalkanoates from Pentoses by Pseudomonas pseudoflava

The potential of Pseudomonas pseudoflava to produce poly-β-hydroxyalkanoates (PHAs) from pentoses was studied. This organism was able to use a hydrolysate from the hemicellulosic fraction of poplar wood as a carbon and energy source for its growth. However, in batch cultures, growth was inhibited completely at hydrolysate concentrations higher than 30% (vol/vol). When P. pseudoflava was grown on the major sugars present in hemicelluloses in batch cultures, poly-β-hydroxybutyric acid (PHB) accumulated when glucose, xylose, or arabinose was the sole carbon source, with the final PHB content varying from 17% (wt/wt) of the biomass dry weight on arabinose to 22% (wt/wt) of the biomass dry weight on glucose and xylose. Specific growth rates were 0.58 h⁻¹ on glucose, 0.13 h⁻¹ on xylose, and 0.10 h⁻¹ on arabinose, while the specific PHB production rates based on total biomass ranged from 0.02 g g⁻¹ h⁻¹ on arabinose to 0.11 g g⁻¹ h⁻¹ on glucose. PHB weight-average molecular weights were 640,000 on arabinose and 1,100,000 on glucose and xylose. The absolute amount of PHB in the cells decreased markedly when nitrogen limitation was relaxed by feeding ammonium sulfate at the end of the PHB accumulation stage of the arabinose and xylose fermentations. Copolymers of β-hydroxybutyric and β-hydroxyvaleric acids were produced when propionic acid was added to shake flasks containing 10 g of glucose liter⁻¹. The β-hydroxyvaleric acid monomer content attained a maximum of 45 mol% when the initial propionic acid concentration was 2 g liter⁻¹.     

New Recombinant Escherichia coli Strain Tailored for the Production of Poly(3-Hydroxybutyrate) from Agroindustrial By-Products

A recombinant E. coli strain (K24K) was constructed and evaluated for poly(3-hydroxybutyrate) (PHB) production from whey and corn steep liquor as main carbon and nitrogen sources. This strain bears the pha biosynthetic genes from Azotobacter sp. strain FA8 expressed from a T5 promoter under the control of the lactose operator. K24K does not produce the lactose repressor, ensuring constitutive expression of genes involved in lactose transport and utilization. PHB was efficiently produced by the recombinant strain grown aerobically in fed-batch cultures in a laboratory scale bioreactor on a semisynthetic medium supplemented with the agroindustrial by-products. After 24 h, cells accumulated PHB to 72.9% of their cell dry weight, reaching a volumetric productivity of 2.13 g PHB per liter per hour. Physical analysis of PHB recovered from the recombinants showed that its molecular weight was similar to that of PHB produced by Azotobacter sp. strain FA8 and higher than that of the polymer from Cupriavidus necator and that its glass transition temperature was approximately 20°C higher than those of PHBs from the natural producer strains.     

Characterization of Thermoanaerobacter Glucose Isomerase in Relation to Saccharidase Synthesis and Development of Single-Step Processes for Sweetener Production

Regulation of glucose isomerase synthesis was studied in Thermoanaerobacter strain B6A, which fermented a wide variety of carbohydrates including glucose, xylose, lactose, starch, and xylan. Glucogenic amylase activities and β-galactosidase were produced constitutively, whereas the synthesis of glucose isomerase was induced by either xylose or xylan. Production of these saccharidase activities was not significantly repressed by the presence of glucose or 2-deoxyglucose in the growth media. Glucose isomerase production was optimized by controlling the culture pH at 5.5 during xylose fermentation. The apparent temperature and pH optima for these cell-bound saccharidase activities were as follows: glucose isomerase, 80°C, pH 7.0 to 7.5; glucogenic amylase, 70°C, pH 5.0 to 5.5; and β-galactosidase, 60°C, pH 6.0 to 6.5 Glucose isomerase, glucogenic amylase, and β-galactosidase were produced in xylose-grown cells that were active and stable at 60 to 70°C and pH 6.0 to 6.5. Under single-step process conditions, these saccharidase activities in whole cells or cell extracts converted starch or lactose directly into fructose mixtures. A total of 96% of initial liquefied starch was converted into a 49:51 mixture of glucose and fructose, whereas 85% of initial lactose was converted into a 40:31:29 mixture of galactose, glucose, and fructose.     

Production and Properties of Xylan-Degrading Enzymes from Cellulomonas uda

Xylan degradation and production of β-xylanase and β-xylosidase activities were studied in cultures of Cellulomonas uda grown on purified xylan from birchwood. β-Xylanase activity was found to be associated with the cells, although in various degrees. The formation of β-xylanase activity was induced by xylotriose and repressed by xylose. β-Xylosidase activity was cell bound. Both constitutive and inducible β-xylosidase activities were suggested. β-Xylanase and β-xylosidase activities were inhibited competitively by xylose. β-Xylanase activity had a pronounced optimum pH of 5.8, whereas the optimum pH of β-xylosidase activity ranged from 5.4 to 6.1. The major products of xylan degradation by a crude preparation of β-xylanase activity, in decreasing order of amount, were xylobiose, xylotriose, xylose, and small amounts of xylotetraose. This pattern suggests that β-xylanase activity secreted by C. uda is of the endosplitting type. Supernatants of cultures grown on cellulose showed not only β-glucanase but also β-xylanase activity. The latter could be attributed to an endo-1,4-β-glucanase activity which had a low β-xylanase activity.     

Construction of a Xylan-Fermenting Yeast Strain through Codisplay of Xylanolytic Enzymes on the Surface of Xylose-Utilizing Saccharomyces cerevisiae Cells

Hemicellulose is one of the major forms of biomass in lignocellulose, and its essential component is xylan. We used a cell surface engineering system based on α-agglutinin to construct a Saccharomyces cerevisiae yeast strain codisplaying two types of xylan-degrading enzymes, namely, xylanase II (XYNII) from Trichoderma reesei QM9414 and β-xylosidase (XylA) from Aspergillus oryzae NiaD300, on the cell surface. In a high-performance liquid chromatography analysis, xylose was detected as the main product of the yeast strain codisplaying XYNII and XylA, while xylobiose and xylotriose were detected as the main products of a yeast strain displaying XYNII on the cell surface. These results indicate that xylan is sequentially hydrolyzed to xylose by the codisplayed XYNII and XylA. In a further step toward achieving the simultaneous saccharification and fermentation of xylan, a xylan-utilizing S. cerevisiae strain was constructed by codisplaying XYNII and XylA and introducing genes for xylose utilization, namely, those encoding xylose reductase and xylitol dehydrogenase from Pichia stipitis and xylulokinase from S. cerevisiae. After 62 h of fermentation, 7.1 g of ethanol per liter was directly produced from birchwood xylan, and the yield in terms of grams of ethanol per gram of carbohydrate consumed was 0.30 g/g. These results demonstrate that the direct conversion of xylan to ethanol is accomplished by the xylan-utilizing S. cerevisiae strain.     

Characterization of a Novel β-Galactosidase from Bifidobacterium adolescentis DSM 20083 Active towards Transgalactooligosaccharides

This paper reports on the effects of both reducing and nonreducing transgalactooligosaccharides (TOS) comprising 2 to 8 residues on the growth of Bifidobacterium adolescentis DSM 20083 and on the production of a novel β-galactosidase (β-Gal II). In cells grown on TOS, in addition to the lactose-degrading β-Gal (β-Gal I), another β-Gal (β-Gal II) was detected and it showed activity towards TOS but not towards lactose. β-Gal II activity was at least 20-fold higher when cells were grown on TOS than when cells were grown on galactose, glucose, and lactose. Subsequently, the enzyme was purified from the cell extract of TOS-grown B. adolescentis by anion-exchange chromatography, adsorption chromatography, and size-exclusion chromatography. β-Gal II has apparent molecular masses of 350 and 89 kDa as judged by size-exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively, indicating that the enzyme is active in vivo as a tetramer. β-Gal II had an optimal activity at pH 6 and was not active below pH 5. Its optimum temperature was 35°C. The enzyme showed highest V_max values towards galactooligosaccharides with a low degree of polymerization. This result is in agreement with the observation that during fermentation of TOS, the di- and trisaccharides were fermented first. β-Gal II was active towards β-galactosyl residues that were 1→4, 1→6, 1→3, and 1↔1 linked, signifying its role in the metabolism of galactooligosaccharides by B. adolescentis.     

Regulation of β-1, 4-Glucosidase Expression by Candida wickerhamii

Candida wickerhamii NRRL Y-2563 expressed β-glucosidase activity (3 to 8 U/ml) constitutively when grown aerobically in complex medium containing either glycerol, succinate, xylose, galactose, or cellobiose as the carbon source. The addition of a high concentration of glucose (>75 g/liter) repressed β-glucosidase expression (<0.3 U/ml); however, this yeast did produce β-glucosidase when the initial glucose concentration was ≤50 g/liter. When grown aerobically in medium containing glucose plus the above-listed carbon sources, diauxic utilization of the carbon source was observed and the expression of β-glucosidase was glucose repressed. Surprisingly, glucose repression did not occur when the cells were grown anaerobically. When grown anaerobically in medium containing 100 g of glucose per liter, C. wickerhamii produced 6 to 9 U of enzyme per ml and did not demonstrate diauxic utilization of glucose-cellobiose mixtures. To our knowledge, this is the first report of apparent derepression of a glucose-repressed enzyme by anaerobiosis.     

Cloning of Protocatechuate 3,4-Dioxygenase Genes from Bradyrhizobium japonicum USDA110

A heterologous gene probe encoding the α and β subunits of the Pseudomonas cepacia protocatechuate 3,4-dioxygenase (PCD) was used to detect its homolog in the genome of Bradyrhizobium japonicum USDA110. Three cosmid clones carrying a 2.2-kb BamHI insert showed high levels of PCD activity. SacI digestion of one of the genomic clones, pBjG17, produced a 2.5-kb insert DNA that complemented a PCD mutant of P. cepacia.     

Heterologous Expression of Lactose- and Galactose-Utilizing Pathways from Lactic Acid Bacteria in Corynebacterium glutamicum for Production of Lysine in Whey

The genetic determinants for lactose utilization from Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 and galactose utilization from Lactococcus lactis subsp. cremoris MG 1363 were heterologously expressed in the lysine-overproducing strain Corynebacterium glutamicum ATCC 21253. The C. glutamicum strains expressing the lactose permease and β-galactosidase genes of L. delbrueckii subsp. bulgaricus exhibited β-galactosidase activity in excess of 1,000 Miller units/ml of cells and were able to grow in medium in which lactose was the sole carbon source. Similarly, C. glutamicum strains containing the lactococcal aldose-1-epimerase, galactokinase, UDP-glucose-1-P-uridylyltransferase, and UDP-galactose-4-epimerase genes in association with the lactose permease and β-galactosidase genes exhibited β-galactosidase levels in excess of 730 Miller units/ml of cells and were able to grow in medium in which galactose was the sole carbon source. When grown in whey-based medium, the engineered C. glutamicum strain produced lysine at concentrations of up to 2 mg/ml, which represented a 10-fold increase over the results obtained with the lactose- and galactose-negative control, C. glutamicum 21253. Despite their increased catabolic flexibility, however, the modified corynebacteria exhibited slower growth rates and plasmid instability.     

Cold-Adapted β-Galactosidase from the Antarctic Psychrophile Pseudoalteromonas haloplanktis

The β-galactosidase from the Antarctic gram-negative bacterium Pseudoalteromonas haloplanktis TAE 79 was purified to homogeneity. The nucleotide sequence and the NH₂-terminal amino acid sequence of the purified enzyme indicate that the β-galactosidase subunit is composed of 1,038 amino acids with a calculated M_r of 118,068. This β-galactosidase shares structural properties with Escherichia coli β-galactosidase (comparable subunit mass, 51% amino sequence identity, conservation of amino acid residues involved in catalysis, similar optimal pH value, and requirement for divalent metal ions) but is characterized by a higher catalytic efficiency on synthetic and natural substrates and by a shift of apparent optimum activity toward low temperatures and lower thermal stability. The enzyme also differs by a higher pI (7.8) and by specific thermodynamic activation parameters. P. haloplanktis β-galactosidase was expressed in E. coli, and the recombinant enzyme displays properties identical to those of the wild-type enzyme. Heat-induced unfolding monitored by intrinsic fluorescence spectroscopy showed lower melting point values for both P. haloplanktis wild-type and recombinant β-galactosidase compared to the mesophilic enzyme. Assays of lactose hydrolysis in milk demonstrate that P. haloplanktis β-galactosidase can outperform the current commercial β-galactosidase from Kluyveromyces marxianus var. lactis, suggesting that the cold-adapted β-galactosidase could be used to hydrolyze lactose in dairy products processed in refrigerated plants.     

Two β-Galactosidases from the Human Isolate Bifidobacterium breve DSM 20213: Molecular Cloning and Expression,Biochemical Characterization and Synthesis of Galacto-Oligosaccharides

Two β-galactosidases, β-gal I and β-gal II, from Bifidobacterium breve DSM 20213, which was isolated from the intestine of an infant, were overexpressed in Escherichia coli with co-expression of the chaperones GroEL/GroES, purified to electrophoretic homogeneity and biochemically characterized. Both β-gal I and β-gal II belong to glycoside hydrolase family 2 and are homodimers with native molecular masses of 220 and 211 kDa, respectively. The optimum pH and temperature for hydrolysis of the two substrates o-nitrophenyl-β-D-galactopyranoside (oNPG) and lactose were determined at pH 7.0 and 50°C for β-gal I, and at pH 6.5 and 55°C for β-gal II, respectively. The k _cat/K _m values for oNPG and lactose hydrolysis are 722 and 7.4 mM⁻¹s⁻¹ for β-gal I, and 543 and 25 mM⁻¹s⁻¹ for β-gal II. Both β-gal I and β-gal II are only moderately inhibited by their reaction products D-galactose and D-glucose. Both enzymes were found to be very well suited for the production of galacto-oligosaccharides with total GOS yields of 33% and 44% of total sugars obtained with β-gal I and β-gal II, respectively. The predominant transgalactosylation products are β-D-Galp-(1→6)-D-Glc (allolactose) and β-D-Galp-(1→3)-D-Lac, accounting together for more than 75% and 65% of the GOS formed by transgalactosylation by β-gal I and β-gal II, respectively, indicating that both enzymes have a propensity to synthesize β-(1→6) and β-(1→3)-linked GOS. The resulting GOS mixtures contained relatively high fractions of allolactose, which results from the fact that glucose is a far better acceptor for galactosyl transfer than galactose and lactose, and intramolecular transgalactosylation contributes significantly to the formation of this disaccharide.     

Heterologous Expression of a Bioactive β-Hexosyltransferase,an Enzyme Producer of Prebiotics,from Sporobolomyces singularis

Galacto-oligosaccharides (GOS) are indigestible dietary fibers that are able to reach the lower gastrointestinal tract to be selectively fermented by health-promoting bacteria. In this report, we describe the heterologous expression of an optimized synthetically produced version of the β-hexosyltransferase gene (Bht) from Sporobolomyces singularis. The Bht gene encodes a glycosyl hydrolase (EC 3.2.1.21) that acts as galactosyltransferase, able to catalyze a one-step conversion of lactose to GOS. Expression of the enzyme in Escherichia coli yielded an inactive insoluble protein, while the methylotrophic yeast Pichia pastoris GS115 produced a bioactive β-hexosyltransferase (rBHT). The enzyme exhibited faster kinetics at pHs between 3.5 and 6 and at temperatures between 40 and 50°C. Enzyme stability improved at temperatures lower than 40°C, and glucose was found to be a competitive inhibitor of enzymatic activity. P. pastoris secreted a fraction of the bioactive rBHT into the fermentation broth, while the majority of the enzyme remained associated with the outer membrane. Both the secreted and the membrane-associated forms were able to efficiently convert lactose to GOS. Additionally, resting cells with membrane-bound enzyme converted 90% of the initial lactose into GOS at 68% yield (g/g) (the maximum theoretical is 75%) with no secondary residual (glucose or galactose) products. This is the first report of a bioactive BHT from S. singularis that has been heterologously expressed.     

Physiological Role of β-Phosphoglucomutase in Lactococcus lactis

A β-phosphoglucomutase (β-PGM) mutant of Lactococcus lactis subsp. lactis ATCC 19435 was constructed using a minimal integration vector and double-crossover recombination. The mutant and the wild-type strain were grown under controlled conditions with different sugars to elucidate the role of β-PGM in carbohydrate catabolism and anabolism. The mutation did not significantly affect growth, product formation, or cell composition when glucose or lactose was used as the carbon source. With maltose or trehalose as the carbon source the wild-type strain had a maximum specific growth rate of 0.5 h⁻¹, while the deletion of β-PGM resulted in a maximum specific growth rate of 0.05 h⁻¹ on maltose and no growth at all on trehalose. Growth of the mutant strain on maltose resulted in smaller amounts of lactate but more formate, acetate, and ethanol, and approximately 1/10 of the maltose was found as β-glucose 1-phosphate in the medium. Furthermore, the β-PGM mutant cells grown on maltose were considerably larger and accumulated polysaccharides which consisted of α-1,4-bound glucose units. When the cells were grown at a low dilution rate in a glucose and maltose mixture, the wild-type strain exhibited a higher carbohydrate content than when grown at higher growth rates, but still this content was lower than that in the β-PGM mutant. In addition, significant differences in the initial metabolism of maltose and trehalose were found, and cell extracts did not digest free trehalose but only trehalose 6-phosphate, which yielded β-glucose 1-phosphate and glucose 6-phosphate. This demonstrates the presence of a novel enzymatic pathway for trehalose different from that of maltose metabolism in L. lactis.     

Galactosyl-Lactose Sialylation Using Trypanosoma cruzi trans-Sialidase as the Biocatalyst and Bovine κ-Casein-Derived Glycomacropeptide as the Donor Substrate

trans-Sialidase (TS) enzymes catalyze the transfer of sialyl (Sia) residues from Sia(α2-3)Gal(β1-x)-glycans (sialo-glycans) to Gal(β1-x)-glycans (asialo-glycans). Aiming to apply this concept for the sialylation of linear and branched (Gal)_nGlc oligosaccharide mixtures (GOS) using bovine κ-casein-derived glycomacropeptide (GMP) as the sialic acid donor, a kinetic study has been carried out with three components of GOS, i.e., 3′-galactosyl-lactose (β3′-GL), 4′-galactosyl-lactose (β4′-GL), and 6′-galactosyl-lactose (β6′-GL). This prebiotic GOS is prepared from lactose by incubation with suitable β-galactosidases, whereas GMP is a side-stream product of the dairy industry. The trans-sialidase from Trypanosoma cruzi (TcTS) was expressed in Escherichia coli and purified. Its temperature and pH optima were determined to be 25°C and pH 5.0, respectively. GMP [sialic acid content, 3.6% (wt/wt); N-acetylneuraminic acid (Neu5Ac), >99%; (α2-3)-linked Neu5Ac, 59%] was found to be an efficient sialyl donor, and up to 95% of the (α2-3)-linked Neu5Ac could be transferred to lactose when a 10-fold excess of this acceptor substrate was used. The products of the TcTS-catalyzed sialylation of β3′-GL, β4′-GL, and β6′-GL, using GMP as the sialic acid donor, were purified, and their structures were elucidated by nuclear magnetic resonance spectroscopy. Monosialylated β3′-GL and β4′-GL contained Neu5Ac connected to the terminal Gal residue; however, in the case of β6′-GL, TcTS was shown to sialylate the 3 position of both the internal and terminal Gal moieties, yielding two different monosialylated products and a disialylated structure. Kinetic analyses showed that TcTS had higher affinity for the GL substrates than lactose, while the V_max and k_cat values were higher in the case of lactose.     

Growth of Azotobacter vinelandii UWD in Fish Peptone Medium and Simplified Extraction of Poly-β-Hydroxybutyrate

Azotobacter vinelandii UWD was grown in a fermentor with glucose medium with and without 0.1% fish peptone (FP) in batch and fed-batch cultures for the production of the natural bioplastic poly-β-hydroxybutyrate (PHB). Strain UWD formed PHB five times faster than cell protein during growth in glucose and NH₄⁺, but PHB synthesis stopped when NH₄⁺ was depleted and nitrogen fixation started. When FP was added to the same medium, PHB accumulated 16 times faster than cell protein, which in turn was inhibited by 40%, and PHB synthesis was unaffected by NH₄⁺ depletion. Thus, FP appeared to be used as a nitrogen source by these nitrogen-fixing cells, which permitted enhanced PHB synthesis, but it was not a general growth stimulator. The addition of FP to the medium led to the production of large, pleomorphic, osmotically sensitive cells that demonstrated impaired growth and partial lysis, with the leakage of DNA into the culture fluid, but these cells were still able to synthesize PHB at elevated rates and efficiency. When FP was continuously present in fed-batch culture, the yield in grams of polymer per gram of glucose consumed was calculated to range from 0.43 g/g, characteristic of nongrowing cells, to an unprecedented 0.65 g/g. Separation of an FP-free growth phase from an FP-containing growth phase in fed-batch culture resulted in better growth of these pleomorphic cells and good production of PHB (yield, 0.32 g/g). The fragility of these cells was exploited in a simple procedure for the extraction of high-molecular-weight PHB. The cells were treated with 1 N aqueous NH₃ (pH 11.4) at 45°C for 10 min. This treatment removed about 10% of the non-PHB mass from the pellet, of which 60 to 77% was protein. The final product consisted of 94% PHB, 2% protein, and 4% nonprotein residual mass. The polymer molecular weight (1.7 × 10⁶ to 2.0 × 10⁶) and dispersity (1.0 to 1.9) were not significantly affected (P = 0.05) by this treatment. In addition, the NH₃ extraction waste could be recycled in the fermentation as a nitrogen source, but it did not promote PHB production like FP. A scheme for improved downstream extraction of PHB as well as the merits of using pleomorphic cells in the production of bioplastics is discussed.     

Isolation, Identification, and Metabolic Role of the Sudanophilic Granules of Zoogloea ramigera

Organisms isolated from activated sludge and identified as Zoogloea ramigera accumulated large amounts of sudanophilic granules as the cultures flocculated. The granules were extracted by chloroform and precipitated with ether from acid-hydrolyzed cells. Identification of the sudanophilic granules as poly-β-hydroxybutyric acid (PHB) was confirmed by physical, chemical, and infrared spectral analyses. The isolated polymer accounted for 12.0 to 50.5% of the dry weight of the cells. The polymer was not synthesized when the culture was grown in a growth-limiting concentration of organic substrate; it did accumulate when the culture was grown in medium enriched with carbon and energy sources. An increase in concentration of intracellular PHB was directly proportional to optical density and uptake of glucose. Aside from intracellular storage of PHB as endogenous metabolite, the accumulation of PHB is noted as a possible mechanism of flocculation.     

Multiple β-Ketothiolases Mediate Poly(β-Hydroxyalkanoate) Copolymer Synthesis in Ralstonia eutropha

Polyhydroxyalkanoates (PHAs) are a class of carbon and energy storage polymers produced by numerous bacteria in response to environmental limitation. The type of polymer produced depends on the carbon sources available, the flexibility of the organism’s intermediary metabolism, and the substrate specificity of the PHA biosynthetic enzymes. Ralstonia eutropha produces both the homopolymer poly-β-hydroxybutyrate (PHB) and, when provided with the appropriate substrate, the copolymer poly(β-hydroxybutyrate-co-β-hydroxyvalerate) (PHBV). A required step in production of the hydroxyvalerate moiety of PHBV is the condensation of acetyl coenzyme A (acetyl-CoA) and propionyl-CoA to form β-ketovaleryl-CoA. This activity has generally been attributed to the β-ketothiolase encoded by R. eutropha phbA. However, we have determined that PhbA does not significantly contribute to catalyzing this condensation reaction. Here we report the cloning and genetic analysis of bktB, which encodes a β-ketothiolase from R. eutropha that is capable of forming β-ketovaleryl-CoA. Genetic analyses determined that BktB is the primary condensation enzyme leading to production of β-hydroxyvalerate derived from propionyl-CoA. We also report an additional β-ketothiolase, designated BktC, that probably serves as a secondary route toward β-hydroxyvalerate production.Polyhydroxyalkanoates (PHAs) are a class of naturally occurring polymers which serve as a carbon and energy reserve in numerous bacterial species. Ralstonia eutropha (formerly designated Alcaligenes eutrophus [41]) produces the homopolymer poly(β-hydroxybutyrate) (PHB) and, when provided with propionate in the feedstock, the copolymer poly(β-hydroxybutyrate-co-β-hydroxyvalerate) (PHBV). R. eutropha is used commercially to produce PHBV, which is a biodegradable thermoplastic.The PHB biosynthetic pathway requires three enzymatic activities: a β-ketothiolase (PhbA), an NADPH-dependent acetoacetyl coenzyme A (acetoacetyl-CoA) reductase (PhbB) and a PHB synthase (PhbC). The first step in production of the homopolymer PHB is catalyzed by β-ketothiolase which condenses two acetyl-CoA molecules to form acetoacetyl-CoA. Formation of the copolymer PHBV requires the additional condensation of acetyl-CoA with propionyl-CoA to form β-ketovaleryl-CoA (Fig. ​(Fig.1).1). Subsequently, the acetoacetyl-CoA and β-ketovaleryl-CoA are converted into a polymer by the activities of the reductase and synthase. The genes encoding these proteins in R. eutropha reside in an operon which has been well characterized (10, 21, 22, 31, 37). Open in a separate windowFIG. 1Pathway for production of PHBV from acetyl-CoA and propionyl-CoA. β-Ketothiolase performs the condensation reactions to generate either acetoacetyl-CoA or β-ketovaleryl-CoA. These are reduced by acetoacetyl-CoA reductase (PhbB) and polymerized by PHB synthase (PhbC).The substrate specificities of these three enzymes are reportedly adequate for production of PHBV copolymer (7–9), but propionate-fed Escherichia coli harboring the R. eutropha phb operon produces essentially PHB homopolymer (35). Moreover, PHBV copolymer can be produced in E. coli after induction of the fatty acid β-oxidation complex, which contains a β-ketothiolase with broad substrate specificity (26, 27, 35). These data suggest that the R. eutropha PHB pathway is capable of producing copolymer, but only in the context of a second β-ketothiolase with broad substrate specificity.R. eutropha is known to produce at least two β-ketothiolases (7), and at least two distinct plasmid clones which express β-ketothiolase have been isolated from R. eutropha (37). In this work, we analyzed the substrate specificity of the PhbA β-ketothiolase and demonstrated that this enzyme catalyzes thiolysis of β-ketovaleryl-CoA very poorly. We determined that R. eutropha expresses at least two β-ketothiolases in addition to PhbA and that these additional enzymes, which we designate BktB and BktC, efficiently utilize β-ketovaleryl-CoA. We also report the isolation and characterization of bktB (β-ketothiolase B), which encodes the BktB β-ketothiolase required for efficient production of PHBV in R. eutropha.     

Compost Grown Agaricus bisporus Lacks the Ability to Degrade and Consume Highly Substituted Xylan Fragments

The fungus Agaricus bisporus is commercially grown for the production of edible mushrooms. This cultivation occurs on compost, but not all of this substrate is consumed by the fungus. To determine why certain fractions remain unused, carbohydrate degrading enzymes, water-extracted from mushroom-grown compost at different stages of mycelium growth and fruiting body formation, were analyzed for their ability to degrade a range of polysaccharides. Mainly endo-xylanase, endo-glucanase, β-xylosidase and β-glucanase activities were determined in the compost extracts obtained during mushroom growth. Interestingly, arabinofuranosidase activity able to remove arabinosyl residues from doubly substituted xylose residues and α-glucuronidase activity were not detected in the compost enzyme extracts. This correlates with the observed accumulation of arabinosyl and glucuronic acid substituents on the xylan backbone in the compost towards the end of the cultivation. Hence, it was concluded that compost grown A. bisporus lacks the ability to degrade and consume highly substituted xylan fragments.