Poly-β-hydroxyalkanoate in Syntrophomonas wolfei

The production of poly--hydroxyalkanoate (PHA) in Syntrophomonas wolfei grown in pure culture or in coculture with Methanospirillum hungatei was studied. PHA was produced by S. wolfei during the exponential phase of growth under both of these cultural conditions. S. wolfei in pure culture also produced PHA in stationary phase when the medium was supplemented with high concentrations of the substrate crotonate. In S. wolfei, PHA levels decreased after growth stopped and most of the substrate was depleted. Altering the C to N ratio of the medium did not affect the amount of PHA made per mg of protein. The incorporation of labeled butyrate but not acetate into PHA during the early stages of growth of S. wolfei and the fact that some of the PHA in a pure culture of S. wolfei grown with trans-2-pentenoate was a polymer of 5-carbon monomer units indicated that a pathway exists for the synthesis of PHA without degradation of the substrate to acetyl-CoA. During the later stages of growth, PHA was made by a pathway which was in equilibrium with the acetate pool. These data indicate that PHA served as a carbon/energy reserve material in S. wolfei, but the synthesis of this polymer was regulated in a manner different from the known pathways.     

Effect of levulinic acid on cell growth and poly-β-hydroxyalkanoate production by Alcaligenes sp. SH-69

Summary Following growth of Alcaligenes sp. SH-69 on glucose as a sole carbon source for the production of poly--hydroxyalkanoates (PHAs), relatively low levels of levulinic acid (LA) were detected. Experiments were carried out in batch and continuous culture, and the effects of LA addition on growth and PHA synthesis were determined. Significant stimulatory effects were observed, greater than those for propionic acid addition. In N-limited two stage continuous culture, a maximal PHA content of 38.3 % (w/w) was achieved with a polyhydroxyvalerate (PHV) content of 23.5 % (molar basis) at D=0.078 l/h. This resulted from the controlled addition of LA at 0.5 g/L/h in the presence of excess glucose.     

Effect of diets containing γ-linolenic acid on n-6 highly unsaturated fatty acid content of rotifer (Brachionus plicatilis)

The effect of various diets containing linoleic and/or -linolenic acids was studied on n-6 fatty acid composition of the rotifer Brachionus plicatilis. The rotifer's abilities for transformations of n-6 fatty acids were evaluated. Diets containing only linolenic acid as n-6 fatty acid induced low levels of other n-6 fatty acids in rotifers while a diet containing also -linolenic acid led to substantial amounts of di homo -linolenic acid in the rotifers through elongation. Desaturation of -linoleic acid to gamma linolenic appears to be the limiting factor of n-6 highly unsaturated fatty acid biosynthesis by the rotifer. Two sets of experiments were compared using different techniques and different sources of -linolenic acid: Spirulina in inert food or borage oil in emulsion with baker's yeast. Rotifers fed with inert diet with Spirulina contained arachidonic acid while those fed with borage oil had very low arachidonic content. High level of n-3 fatty acids incorporated into the diets seemed to exert inhibitory effects on n-6 transformation rate.     

β-hydroxypropionic acid production by Byssochlamys sp. grown on acrylic acid

A strain of Byssochlamys sp. produced -hydroxypropionic acid when grown on media containing high concentrations of acrylic acid. The maximal production of -hydroxypropionic acid was 4.8% when the initial culture medium contained 7% acrylic acid and 2% glucose, and the initial culture pH was adjusted to 7.0. -Hydroxypropionic acid production from acrylic acid depended greatly on the pH of the culture medium. Calcium hydroxide was the best neutralizer. Correspondence to: K. Takamizawa     

Role of poly-β-hydroxybutyric acid in cyst formation byAzotobacter

Several parameters associated with the growth ofAzotobacter vinelandii in liquid culture were examined in order to investigate the relationship between the accumulation and degradation of poly-β-hydroxybutyric acid (PHB), the development of viscous capsular components, and cyst formation. The amount of intracellular PHB, which increased markedly during the log phase of growth, reached a maximum during the early stationary phase and subsequently declined. During polymer degradation there was a concurrent increase in the extent of encystment in the cultures supplemented with CaCO₃. An increase was noted in the viscosity of culture supernatants during polymer degradation when CaCO₃ was deleted from the medium and the culture pH was controlled by the periodic addition of 0.1m KOH. The extent of encystment and the amount of PHB accumulated were directly proportional to the substrate concentration. The PHB was selectively labeled by the addition of sodium acetate-2-¹⁴C to late log-phase cells. During polymer utilization in either encysting or nonencysting cultures 20% of the label was evolved as CO₂. In the nonencysting cultures, 45% of the radioactivity was distributed between residual PHB and other cellular components, and 35% was in the supernatant polysaccharide-like material. Intact cysts retained 80% of the label. Experiments with ruptured cysts indicated that about 35% of the radioactivity was present in the intine material.     

The isolation of mutants not accumulating poly-β-hydroxybutyric acid

Summary Five mutant strains of Hydrogenomonas H 16 which synthesize poly--hydroxybutyric acid either slowly or not at all have been isolated following nitrite and NMG treatment of wild type cells. When grown on a nitrogen deficient agar medium, the colonies of PHB-free cells can be recognized by their diminished retention of the dye sudanblack B. Enrichment procedures for such mutants have been devised employing the ³²P-phosphate inactivation technique and sucrose gradient centrifugation. The mutants have been characterized with respect to their growth properties, respiratory control and other properties.     

Free fatty acid receptor 4-β-arrestin 2 pathway mediates the effects of different classes of unsaturated fatty acids in osteoclasts and osteoblasts

Bone is a dynamic tissue that is constantly remodelled by bone resorbing osteoclasts and bone forming osteoblasts, respectively. A breakdown in the remodelling process underlies several bone diseases such as osteoporosis. Unsaturated fatty acids (UFAs) have been shown to have beneficial effects on bone health. However, the mechanism of action of UFAs in bone remains unclear. Free fatty acid receptor 4 (FFAR4) is expressed in bone cells and preferentially binds ω−3 and ω−7 UFAs. Therefore, we sought to determine if FFAR4 influenced the action of different classes of UFAs in bone cells. FFAR4 and potential signalling pathways, β-arrestin 2 (βarr2) and G_αq, were silenced in RAW264.7 murine macrophages (pre-osteoclasts) and MC3T3-E1 murine pre-osteoblasts. Cell differentiation, activation of signalling pathways and expression of regulatory genes were evaluated. The ω−3 UFAs, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), and the ω−7 UFA, palmitoleic acid (PLA), were shown to require the FFAR4/βarr2 signalling pathway to inhibit osteoclast differentiation in RAW264.7 murine macrophages. The ω−6 UFA, arachidonic acid, and the ω−9 UFA, oleic acid (OA), were shown to inhibit osteoclast formation but did not use FFAR4. DHA, EPA, PLA and OA enhanced osteoblast signalling through the FFAR4/βarr2 signalling axis. This study reveals that FFAR4/βarr2 signalling may mediate the bone protective effects of different classes of UFAs in osteoclasts and osteoblasts.     

Production of poly-β-hydroxybutyric acid from carbon dioxide by Alcaligenes eutrophus ATCC 17697T

The characteristics of PHB production from carbon dioxide by autotrophic culture of Alcaligenes eutrophus ATCC 17697^T using a recycled gas closed circuit culture system under the condition of oxygen limitation were investigated. Cell concentration increased to more than 60 g/l after 60 h of cultivation, while the PHB concentration reached 36 g/l. PHB accumulation in the oxygen-limited culture was superior than that in an ammonium-deficient culture. The PHB produced was identified as a homopolymer of d-3-hydroxybutyrate by ¹H and ¹³C NMR analysis. The stoichiometry for PHB production from CO₂ under the oxygen limitation condition was indicated to be as follows: 33H₂ + 12O₂ + 4CO₂ → C₄H₆O₂ + 30H₂O. This stoichiometry shows that the hydrogen consumption per one mole of CO₂ for PHB production is larger than that for cell formation.     

Kinetics and effect of nitrogen source feeding on production of poly-β-hydroxybutyric acid by fed-batch culture

Summary A kinetic study of the production of poly--hydroxybutyric acid (PHB) by a fed-batch culture of Protomonas extorquens showed that a nitrogen source was necessary even in the PHB production phase. The effect of ammonia feeding on PHB production was consequently investigated. The nitrogen source (ammonia water) was supplied at a low constant feeding rate after the growth phase in which cell mass concentration reached 60 g/l. Feeding with a small quantity of ammonia resulted in a more rapid increase in intracellular PHB content than was the case without ammonia feeding. Excessive feeding of ammonia, however, caused not only degradation of accumulated PHB but also reduction of microbial PHB synthetic activity.     

Regulating effect of β-ketoacyl synthase domain of fatty acid synthase on fatty acyl chain length in de novo fatty acid synthesis

Fatty acid synthase (FAS) is a multifunctional homodimeric protein, and is the key enzyme required for the anabolic conversion of dietary carbohydrates to fatty acids. FAS synthesizes long-chain fatty acids from three substrates: acetyl-CoA as a primer, malonyl-CoA as a 2 carbon donor, and NADPH for reduction. The entire reaction is composed of numerous sequential steps, each catalyzed by a specific functional domain of the enzyme. FAS comprises seven different functional domains, among which the β-ketoacyl synthase (KS) domain carries out the key condensation reaction to elongate the length of fatty acid chain. Acyl tail length controlled fatty acid synthesis in eukaryotes is a classic example of how a chain building multienzyme works. Different hypotheses have been put forward to explain how those sub-units of FAS are orchestrated to produce fatty acids with proper molecular weight. In the present study, molecular dynamic simulation based binding free energy calculation and access tunnels analysis showed that the C16 acyl tail fatty acid, the major product of FAS, fits to the active site on KS domain better than any other substrates. These simulations supported a new hypothesis about the mechanism of fatty acid production ratio: the geometric shape of active site on KS domain might play a determinate role.     

Cytotoxic aggregates of α-lactalbumin induced by unsaturated fatty acid induce apoptosis in tumor cells

The effects of three fatty acids on cytotoxic aggregate formation of Ca²⁺-depleted bovine α-lactalbumin (apo-BLA) have been studied by UV absorbance spectroscopy and transmission electron microscopy. The experimental results demonstrate that two unsaturated fatty acids, oleic acid and linoleic acid, and one saturated fatty acid, stearic acid, induce the intermediate of apo-BLA at pH 4.0-4.5 to form amorphous aggregates in time- and concentration-dependent manners. These aggregates are dissolved under physiological conditions at 37 °C and further characterized by fluorescence spectroscopy, circular dichroism and time-of-flight mass spectrometry. Our data here indicate that the structural characteristics of these aggregates are similar to those of HAMLET/BAMLET (human/bovine α-lactalbumin made lethal to tumor cells), a complex of the partially unfolded α-lactalbumin with oleic acid. Cell viability experiments indicate the aggregates of apo-BLA induced by oleic acid and linoleic acid show significant dose-dependent cytotoxicity to human lung tumor cells of A549 but those induced by stearic acid have no toxicity to tumor cells. Furthermore, the cytotoxic aggregates of apo-BLA induced by both unsaturated fatty acids induce apoptosis of human lung cancer cell line A549, suggesting that such cytotoxic aggregates of apo-BLA could be potential antitumor drugs. The present study provides insight into the mechanism of fatty acid-dependent oligomerization and cytotoxicity of α-lactalbumin, and will be helpful in the understanding of the molecular mechanism of HAMLET/BAMLET formation.     

Carotenol fatty acid esters: easy substrates for digestive enzymes?

To study the specificity of gastric lipases on carotenoid mono- and diesters, an enzymatic assay was applied. Digestions were carried out in phosphate buffer at pH 7.4 and 37 °C. As substrates we employed oleoresins from marigold (Tagetes erecta L.; lutein diesters), red paprika (Capsicum annuum L., mainly capsanthin diesters), papaya (Carica papaya L.; β-cryptoxanthin esters), and loquat (Eriobotrya japonica Lindl.; β-cryptoxanthin esters) as well as retinyl palmitate. These were reacted with porcine pancreatic lipase, porcine pancreatin, porcine cholesterol esterase, and human pancreatic lipase. As reference enzyme a yeast lipase from Candida rugosa was applied. A high turnover could be observed with porcine pancreatic lipase and porcine cholesterol esterase, indicating cholesterol esterase to be a plausible candidate for generation of free carotenoids in the gut. Human pancreatic lipase accepted only retinyl palmitate as substrate, carotenoid mono- and diesters were not hydrolyzed. The assay permits an approach for calculation of enzymatic activities towards carotenoid esters as substrates for the first time, which is based on the amount of enzyme formulation, present in the assay (U/mg solid). Furthermore, these studies provide deeper insight into carotenoid ester bioaccessibility.     

Mass production of poly-β-hydroxybutyric acid by fully automatic fed-batch culture of methylotroph

Summary Fifty-one methylotrophs were checked with respect to their ability of poly--hydroxybutyric acid (PHB) production from methanol. One of them, Pseudomonas sp. K, was chosen from its good growth on a minimum synthetic medium. Optimal temperature and pH for its growth were 30° C and 7.0, respectively. Concentrations of PO ₄ ^3- and NH ₄ ⁺ in the medium should be kept at low levels. PHB formation was stimulated by deficiency of nutrient such as NH ₄ ⁺ , SO ₄ ^2- , Mg²⁺, Fe²⁺ or Mn²⁺. Among them, nitrogen deficiency was chosen from its effectiveness and easiness for PHB accumulation.The microorganism was cultivated to produce a large amount of poly--hydroxybutyric acid (PHB) from methanol by means of microcomputer-aided fully automatic fed-batch culture technique. During the cultivation, temperature, dissolved oxygen concentration (DO), and methanol concentration in the culture broth were maintained at 30° C 2.5±0.5 ppm and 0.5±0.2 g/l, respectively. Other nutrients, nitrogen source and mineral ions, were also controlled to maintain their initial concentrations in the medium during cell growth phase. When the high cell concentration was achieved (160 g/l), feedings of ammonia and minerals were stopped and only methanol was supplied successively to accumulate PHB. At 175 h, high concentration of PHB (136 g/l) was obtained and total cell concentration became 206 g/l. DO must be maintained above the critical level during the PHB formation phase, too. PHB yield from methanol (g PHB/g methanol) was 0.18 and the maximum PHB content reached 66% of dry weight. Solid PHB produced by the strain had the melting point of 176° C and the average molecular weight of 3.0x10⁵.     

Immunomodulatory effect of poly-γ-glutamic acid derived from Bacillus subtilis on natural killer dendritic cells

Bacillus subtilis-derived poly-γ-glutamic acid (γPGA) stimulates dendritic cells (DCs) to produce IL12, leading to CD4⁺ T cell differentiation toward the Th1 phenotype, but DCs consist of heterogeneous subpopulations with a variety of immune functions. Among these, natural killer dendritic cells (NKDCs) play an important role in anti-tumor immune responses. Herein, we demonstrate the role of NKDCs in γPGA-meditated anti-tumor immune responses. NK1.1⁺ CD11c⁺ NKDCs were stimulated upon γPGA stimulation in vitro and in vivo to up-regulate lymphocyte activation markers, MHC class I and II, and co-stimulatory molecules. In particular, NKDCs were activated by γPGA to produce IFNγ and TNFα, like NK cells, as well as IL12, like DCs, implying that NKDCs have unique and multifunctional roles. Importantly, NKDCs stimulated by γPGA conferred stronger anti-tumor effects in mice and showed increased cytotoxicity against various tumor cell lines in vitro. In conclusion, NKDCs are one of the key players in anti-tumor immunity induced by γPGA.     

Effect of 1-amino-4-sulphonate-β-naphthol on the oil content and fatty acid composition of peanut

Exogenously applied 1-amino-4-sulphonate-β-naphthol increased the oil content and altered the fatty acid composition of the semispreading variety C-501 of peanut. The content of oleic acid decreased while that oflinoleic acid increased in the kernels of treated plants.     

Manipulation of the genes for poly-β-hydroxybutyric acid synthesis in Alcaligenes eutrophus

Summary In order to establish the molecular breeding system in Alcaligenes eutrophus producing poly--hydroxybutyric acid (PHB), phbCAB genes from A. eutrophus were recombined into the E. coli-A. eutrophus shuttle vector and directly transferred into A. eutrophus by the electroporation. In A. eutrophus transformants, recombinant plasmids were stably maintained and enzyme activities for PHB biosyntheses were elevated 1.4–2.7 fold by the cloned genes.     

High frequency transformation of Alcaligenes eutrophus producing poly-β-hydroxybutyric acid by electroporation

Summary A strain of Alcaligenes eutrophus producing poly--hydroxybutyric acid was successfully transformed by the electroporation. The plasmid used was a broad host range plasmid pKT230 conferring kanamycin resistance. The optimum yield of transformant was 0.8×10²/g DNA when 50 l competent cells at 10¹⁰/ml were pulsed by 11.5 kV/cm for 5 ms with 1 g DNA. Plasmid DNA in the A. eutrophus transformant was stably maintained as a monomeric structure.     

Production of poly(β-l-malic acid) (PMA) from agricultural biomass substrates by Aureobasidium pullulans

For the first time the production of poly(β-l -malic acid) (PMA) has been achieved using agricultural biomass substrates by the yeast-like fungus Aureobasidium pullulans. Strains NRRL Y-2311-1, NRRL 50382, NRRL 50383, and NRRL 50384, representing diverse isolation sources and phylogenetic clades, produced PMA from alkaline H₂O₂-pretreated corn fiber and wheat straw as sole carbon sources. Pretreated wheat straw was better than pretreated corn fiber, and strain NRRL 50383 gave the highest overall yields of PMA. The addition of CaCO₃ plus supplementary hydrolytic enzymes enhanced PMA production. Four basal media were compared for PMA production, and the best was found to be a N-limited pullulan production medium (PM). In this medium, PMA production took place during growth limitation. Under optimal conditions, strain NRRL 50383 produced more than 20 g PMA/l from 5 % (w/v) pretreated wheat straw in PM with 3 % (w/v) CaCO₃ and supplementary enzymes.     

Synthesis of α-ketoglutaric acid by Yarrowia lipolytica yeast grown on ethanol

The ability of yeast to synthesize α-ketoglutaric acid (KGA) from ethanol has been studied. Thiamine-auxotrophic yeasts of different genera and species may be able to produce KGA; the main condition of synthesis is growth limitation by thiamine. Using a model culture, mutant Yarrowia lipolytica N 1, the principal conditions affecting KGA oversynthesis were identified. These were: thiamine concentration in medium and in cells, nitrogen and oxygen concentration in medium, and pH level. A KGA concentration of 49 g/l and a yield from ethanol consumed of 42% were achieved. Based on the results of the analysis of the activities of the key enzymes participating in ethanol metabolism and KGA synthesis, a concept of the mechanism of KGA biosynthesis by Y. lipolytica yeast is suggested and discussed. Received: 1 March 1999 / Received revision: 28 June 1999 / Accepted: 5 June 1999