Anthocyanin synthesis in a white flowering mutant of Petunia hybrida

In flower buds of the white flowering mutant W19 of Petunia hybrida four biologically active dihydroflavonol intermediates-dihydroquercetin-7-glucoside, dihydroquercetin-4-glucoside, dihydroquercetin, and dihydrokaempferol-7-glucoside-are accumulated. When dihydroquercetin was supplied to in vitro cultured corollas of the white flowering mutant W18, a mixture of cyanidin and delphinidin glycosides was produced, cyanidin-3-glucoside being the major pigment. The quantity of dihydroquercetin accumulated in W19 is very small, but this compound appears to be a more direct precursor of anthocyanins than the glucosides of dihydrokaempferol and dihydroquercetin. The conditions for pigment synthesis in W18 were optimalized. The quantitative uptake of dihydroquercetin was also studied. It was demonstrated that ca. 1/3 of the quantity present in the culture solution entered the corolla. From the absorbed dihydroquercetin only 14% was converted into anthocyanins. Complementation experiments to determine the biosynthetic sequence of the anthocyanin genes An1, An2, and An3 indicated that the genes An1 and An2 are indistinguishable by this technique.Abbreviation DHQ (+) dihydroquercetin     

Degradation of 4-aminobenzenesulfonate by a two-species bacterial coculture

The mutualistic interactions in a 4-aminobenzenesulfonate (sulfanilate) degrading mixed bacterial culture were studied. This coculture consisted of Hydrogenophaga palleronii strain S1 and Agrobacterium radiobacter strain S2. In this coculture only strain S1 desaminated sulfanilate to catechol-4-sulfonate, which did not accumulate in the medium but served as growth substrate for strain S2. During growth in batch culture with sulfanilate as sole source of carbon, energy, nitrogen and sulfur, the relative cell numbers (colony forming units) of both strains were almost constant. None of the strains reached a cell number which was more than threefold higher than the cell number of the second strain. A mineral medium with sulfanilate was inoculated with different relative cell numbers of both strains (relative number of colony forming units S1:S2 2200:1 to 1:500). In all cases, growth was found and the proportion of both strains moved towards an about equal value of about 3:1 (strain S1:strain S2). In contrast to the coculture, strain S1 did not grow in a mineral medium in axenic culture with 4-aminobenzenesulfonate or any other simple organic compound tested. A sterile culture supernatant from strain S2 enabled strain S1 to grow with 4-aminobenzenesulfonate. The same growth promoting effect was found after the addition of a combination of 4-aminobenzoate, biotin and vitamin B₁₂. Strain S1 grew with 4-aminobenzenesulfonate plus the three vitamins with about the same growth rate as the mixed culture in a mineral medium. When (resting) cells of strain S1 were incubated in a pure mineral medium with sulfanilate, up to 30% of the oxidized sulfanilate accumulated as catechol-4-sulfonate in the culture medium. In contrast, only minor amounts of catechol-4-sulfonate accumulated when strain S1 was grown with 4ABS in the presence of the vitamins.Abbreviations 4ABS 4-aminobenzenesulfonate - CFU colony forming units - 4CS catechol-4-sulfonate - 4HB 4-hydroxybenzoate     

Alkaloid production in cell suspension cultures of Thalictrum flavum and T. dipterocarpum

Both cell suspension cultures of Thalictrum flavum and T. dipterocarpum were found to produce berberine (0.3 and 0.4 g/l, respectively) as a main alkaloid. Berberine production in the latter was markedly stimulated by 1-naphthaleneacetic acid in combination with 6-benzylaminopurine, whereas it was rather suppressed by the same auxin in the former. T. flavum cultures accumulated berberine and columbamine in the cells without releasing them into medium. On the other hand, T. dipterocarpum cultures released berberine into medium during the logarithmic growth phase, but thereafter accumulated all the berberine synthesized in the cells.Abbreviations LS Linsmaier and Skoog - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - BA 6-benzylaminopurine - TFG a culture strain of T. flavum ssp. glaucum - TDP a culture strain of T. dipterocarpum     

Improving NADPH availability for natural product biosynthesis in Escherichia coli by metabolic engineering

With microbial production becoming the primary choice for natural product synthesis, increasing precursor and cofactor availability has become a chief hurdle for the generation of efficient production platforms. As such, we employed a stoichiometric-based model to identify combinations of gene knockouts for improving NADPH availability in Escherichia coli. Specifically, two different model objectives were used to identify possible genotypes that exhibited either improved overall NADPH production or an improved flux through an artificial reaction coupling NADPH yield to biomass. The top single, double and triple gene deletion candidates were constructed and as a case study evaluated for their ability to produce two polyphenols, leucocyanidin and (+)-catechin. Each is derived from their common precursor dihydroquercetin using two recombinant NADPH-dependent enzymes: dihydroflavonol 4-reductase and leucoanthocyanidin reductase. The best engineered strain carrying Δpgi, Δppc and ΔpldA deletions accumulated up to 817 mg/L of leucocyanidin and 39 mg/L (+)-catechin in batch culture with 10 g/L glucose in modified M9 medium, a 4-fold and 2-fold increase, respectively, compared to the wild-type control.     

Utilization of an alternative carbon source for efficient production of human alpha(1)-antitrypsin by genetically engineered rice cell culture

Human alpha(1)-antitrypsin was produced by genetically engineered rice cells using promoter and signal peptide of a rice alpha-amylase isozyme. Batch and continuous cultures were employed to investigate the effects of alternative carbon sources on the alpha(1)-antitrypsin production. While this expression system is inducible by sugar depletion, we have found that the productivity of alpha(1)-antitrypsin increased 2.4- to 3.4-fold, compared with the control medium without carbon source, in medium containing an alternative carbon source, such as pyruvic acid and glyoxylic acid. The accumulated alpha(1)-antitrypsin in the medium containing pyruvic acid reached 18.2-24.2 mg/g-dry cell in 50-70 h by batch culture.     

Glutamine uptake and utilization by preimplantation mouse embryos in CZB medium

At least 71% of CF1 x B6SJLF1/J embryos developed from the 1-cell stage to the blastocyst stage in an optimum glutamine concentration of 1 mM, as long as glucose was present after the first 48 h of culture. Blastocysts raised under these conditions had significantly more cells than did blastocysts raised in CZB medium alone (glutamine present, glucose absent). Embryos raised in vivo accumulated 170-200 fmol glutamine/embryo/h at the unfertilized egg and 1-cell stages with a decline to 145 fmol/embryo/h at the 2-cell stage, followed by sharp increases to 400 and 850 fmol/embryo/h at the 8-cell and blastocyst stages. The presence or absence of glucose in the labelling medium had no effect on glutamine uptake by these embryos. Embryos raised in vitro accumulated 2-3 times more glutamine at stages comparable to those of embryos raised in vivo. In all cases in which 1-cell to blastocyst development in vitro was successful, glucose was present in the culture medium and the incremental uptake of glutamine between the 8-cell stage and the blastocyst stage was approximately 2-fold. This was also the increment for in-vivo raised embryos. When glucose was not present after the first 48 h, the 8-cell to blastocyst glutamine increment was not significant, and development into blastocysts was reduced. The results also show that glutamine can be used as an energy source for the generation of CO2 through the TCA cycle by all stages of preimplantation mouse development, whether raised in vivo or in vitro from the 1-cell stage. Two-cell embryos raised in vivo converted as much as 70% of the glutamine uptake into CO2, consistent with an important role for glutamine in the very earliest stages of preimplantation development. Cultured blastocysts appeared to convert less glutamine and the presence of glucose in the culture medium seemed to inhibit this conversion.     

Trehalose accumulation from soluble starch by Saccharomycopsis fibuligera sdu

Trehalose accumulation from starch by Saccharomycopsis fibuligera sdu was examined in 300-ml shaken flask culture and Biostat B(2) 2-1 fermentation. In the 300-ml flask, 16.5% (w/w) trehalose accumulated in the yeast cells (cell dry weight) was observed with 100-ml medium shaken at 200 rpm for 50 h at 30 degrees C. We found that 1.0% soluble starch in the medium was most suitable for trehalose accumulation by this yeast strain. In the Biostat B(2) 2-1 fermentor, 18.0% (w/w) trehalose accumulated in the yeast cells (cell dry weight) was observed within 48 h of fermentation when agitation speed was 200 rpm. The trehalose obtained from the yeast cells was identical to standard trehalose from Sigma based on the analysis results of High-Performance Exchange Anionic Chromatography (HPEAC).     

Chemical potential of Aphelandra sp. cell cultures

Six different callus lines and three different suspension culture lines were established from plants of two Aphelandra species (Acanthaceae). All established lines were analyzed for secondary metabolite accumulation. A discrepancy between secondary metabolites accumulated in the plants and in the cell cultures could be observed. All established Aphelandrasp. cell cultures produced verbascoside (acteoside) as the major extractable metabolite. Time course experiments were carried out to investigate the relationship between cell growth and verbascoside production. In the present study it was shown that verbascoside accumulation was growth dependent and positively related to the presence of 2,4-D in the medium. The conditions in which verbascoside represents ca. 18% of cell culture weight have been defined. Free polyamines were detected in the cell culture lines cultivated in MS liquid medium (cysteine 10 mg l^-1, thiamine 1 mg l^-1, 2,4-D 1 mg l^-1, kinetin 0.2 mg l^-1 and sucrose 30 g l^-1). Putrescine and spermidine accumulated within 8 days to a maximum of 8.4 μmol g^-1 of dry wt and 2.6 μmol g^-1 of dry wt respectively and thereafter their concentration decreased rapidly. There was no evidence for the presence of spermine or any other type of free or conjugated polyamines in the tested cell culture lines. This revised version was published online in June 2006 with corrections to the Cover Date.     

Overproduction of thymidine by recombinant Brevibacterium helvolum amplified with thymidine monophosphate phosphohydrolase gene from bacteriophage PBS2

A microbial fermentation process could be used to produce thymidine biologically but many of the enzymes related to nucleotide biosynthesis are highly regulated. To overcome the complex regulation steps, an analogue mutant of Brevibacterium helvolum resistant to fluorouracil, hydroxyurea, and trimethoprim was constructed. This mutant accumulated 380 mg thymidine 1(-1) in 16 h in shake-flask culture. However, the accumulation of thymidine monophosphate (TMP) inside the cells suggested a low activity of nucleotidase which degrades TMP to thymidine. This limitation was overcome by cloning the TMP phosphohydrolase (TMPase) gene of the unusual bacteriophage, PBS2. As a result, TMP in recombinant cells decreased from 230 micromol g(-1) cell to 20 micromol g(-1) cell with accumulation of 500 mg thymidine l(-1) in the medium.     

Intracellular Changes in Ions and Organic Solutes in Halotolerant Brevibacterium sp. Strain JCM 6894 after Exposure to Hyperosmotic Shock

In the present study we aimed to observe the intracellular responses when there was a hyperosmotic shock with a large shift in ionic strength in nutrient-rich and nutrient-poor external environments in order to clarify the availability of substrates. To do this, we used the halotolerant organism Brevibacterium sp. strain JCM 6894, which is able to grow in the presence of a wide range of salt concentrations. Hyperosmotic shock was induced by transferring cells in the late exponential phase of growth in a complex medium containing 0.5 M NaCl into either old or fresh culture medium containing 2 M NaCl. Changes in the growth rate, in the pH of the medium, and in the internal cation or organic solute concentrations in the cytosol after an upshock were analyzed as a function of incubation time. The cells exhibited very different responses to upshocks in fresh culture medium and in old culture medium; in fresh culture medium, growth was stimulated and the medium became more acidic, whereas the old culture medium repressed growth and the medium became more alkaline. The intracellular free Na⁺ concentrations remained low (80 nmol mg of protein⁻¹) after an upshock in fresh culture medium, although they quickly increased twofold in the old culture medium. In contrast, K⁺ ions immediately accumulated in the cells in fresh culture medium, whereas K⁺ ions were taken up quite slowly in old culture medium. Furthermore, the cells placed in fresh culture medium transiently accumulated alanine and glutamine in response to the upshock, but the cells placed in old culture medium did not. Growth of the Brevibacterium strain at higher levels of salinity was supported by ectoine synthesis but was not observed after the shift to high-osmolarity conditions in the old culture. In the fresh culture, however, ectoine was vigorously synthesized in cells for more than 5 h after the upshock; the concentration of ectoine in cells was more than 3,500 nmol mg of protein⁻¹ at 10 h, which corresponded to a ninefold increase compared to the concentration before the shock. These findings are consistent with the results of an analysis of the extracellular medium composition before and after the upshock.     

Enhanced production of scopolin bySolanum aviculare cells immobilised within Ca-alginate gel beads

Summary Different matrices, obtained by varying calcium (0.1 to 1.5M) and alginate (1 to 1.5%) concentrations, were used to study the influence of immobilisation parameters on the behaviour ofS. aviculare. A significant modulation of cell growth, cell release, and scopolin production and excretion has been observed. Physiological and morphological characteristics ofSolanum aviculare cells immobilised within Ca-alginate beads were notably different from those of suspended cells. ImmobilisedS. aviculare have accumulated scopolin (up to 120 g·g^–1 FWB) within beads and excreted it into the culture medium (up to 8 g·g^–1 FWB). Contrary to suspended cells which have accumulated only traces of this metabolite within intracellular compartments (1 g·g^–1 FWB), no scopolin has been found into the culture medium.Abbreviations ANA -Naphthaleneacetic acid - HPLC high performance liquid chromatography - FWB fresh weight biomass - LS medium Linsmaier and Skoog medium - MS mass spectroscopy - NMR nuclear magnetic resonance - r² coefficient of determination - s standard deviation     

Cholesterol synthesis by several strains of Escherichia]

The results of the study of lipids in the media used for growing different Escherichia strains are presented. Donor plasma with carbon-labeled sodium acetate was used as culture medium. Those strains which induced an increase in cholesterol content in the medium after 48-hour incubation were considered cholesterin-synthetizing. During the growth of these strains the radioactive marker became incorporated into the lipids accumulated in the medium: phospholipids, cholesterol, fatty acids. The degree of this incorporation depended on the dose of labeled sodium acetate and the amount of the inoculated culture. Cholesterol-synthetizing activity of Escherichia is characteristic of only freshly isolated strains.     

Phenotypic differences in anthocyanin accumulation among clonally related cultured cells of carrot

Subclones from a wild carrot cell culture have been examined for their anthocyanin accumulation in the absence and presence of DMSO and 4-coumaric acid, naringenin, dihydroquercetin or leucocyanidin. Subclones that accumulate no or extremely low levels of anthocyanin do not increase their anthocyanin accumulation when treated with DMSO or intermediates. These compounds increased the anthocyanin accumulation in subclones which produce detectable anthocyanin in their absence.Chalcone synthase was shown to be present in clones and the activity showed no correlation with the amount of anthocyanin accumulated. This suggests that the enzymes of anthocyanin biosynthesis are not coordinately repressed in the subclones which accumulate little or no anthocyanin. Dihydroquercetin and catechin were present in subclones with little or no anthocyanin but no procyanidin was detected which suggests that these subclones biosynthesize leucocyanidin but do not convert it into colorless procyanidins as a major alternative metabolic pathway to anthocyanin biosynthesis. The possibility that some clones are not anthocyanin accumulating because they have impaired transport of the sinapoylated anthocyanin into the vacuole is discussed.     

二氢槲皮素的研究进展

二氢槲皮素是自然界中一种重要的黄酮类化合物，主要存在于高寒带落叶松的根部。由于其具有较好的抗氧化、抗肿瘤等生物学活性而被广泛应用于食品领域、工业领域和医药领域。然而，目前二氢槲皮素的工业化生产仍然依赖于传统的植物提取，原料稀缺、提取难度大、产率较低，阻碍了其工业化应用的推进。基于此，主要综述了二氢槲皮素的化学结构及性质、生物合成的分子机制、生物学活性以及生产工艺的研究进展，并对未来二氢槲皮素的相关研究趋势进行了展望，以期为日后二氢槲皮素的生物合成研究提供理论参考。     

Oxidation of lecithin in the presence of dihydroquercetin and its complex with divalent iron ions

The ability of the flavonoid dihydroquercetin to prevent or accelerate the accumulation of reactive oxygen species and the metabolites of oxidative stress, carbonyl compounds has been studied. It has been shown on a model of oxidation of lecithin that dihydroquercetin exhibits a prooxidant effect in the alkaline region of pH, whereas at neutral and acidic pH values dihydroquercetin is an effective antioxidant. In the presence of ferrous iron ions, which catalyze the Fenton reaction, dihydroquercetin forms a complex with metal that shows the antioxidant activity in the region of high pH values. It has been found that the oxidation of lecithin in the presence of 20–200 μM ferrous iron is inhibited by dihydroquercetin to a concentration of 3.2 mM. At higher concentration of dihydroquercetin in the presence of ferrous iron, accumulation of malonic dialdehyde occurs, indicating the presence of the prooxidant activity of dihydroquercetin.     

Newly developed primary culture of rat visceral adipocytes and their in vitro characteristics

We have recently developed a primary culture system for visceral adipocytes (VAs) using stomal-vascular cells (SVCs) isolated from the mesenteric fat tissue of male Sprague-Dawley rats of 3-5 weeks of age. Modified Dulbecco's modified Eagle medium (DMEM)/F12 containing 17 microM pantothenic acid, 33 microM biotin, 100 microM ascorbic acid, 1 microM octanoic acid, 50 nM triiodothyronine, 10 microg/ml insulin, 10% newborn calf serum (NCS), 100 units/ml penicillin and 100 microg/ml streptomycin was used as a basal culture medium, which did not contain any synthetic compounds usually used to promote adipogenesis, such as indomethacin, dexamethasone, or peroxisome proliferator-activated receptor (PPAR)-gamma agonists. The SVCs differentiated and proliferated efficiently, and formed a confluent monolayer in 3 days. The VAs accumulated lipids droplets in their cytoplasm at approximately 7 days. The differentiation rate from applied SVCs to mature adipocytes was >80% per culture. Adiponectin concentration in the medium increased from Day 5 to Day 7. Application of lipid emulsion stimulated maturation of the SVCs into VAs, as well as subsequent lipid accumulation. Norepinephrine (2 x 10(-5) mM) reduced the size of lipid particles and decreased triglyceride (TG) content in the matured adipocytes at 30 min. These results indicate that the new culture system is sufficient to maintain the physiological activity of visceral adipose tissue similar to that in vivo, making it an appropriate and useful tool for basic and applied research on obesity.     

Effect of the medium composition on the accumulation of 2-keto-D-gluconic acid in Pseudomonas putida cultures

The effect of the composition of the culture medium and the age of the culture on the activities of the enzymes involved in accumulation of 2-ketogluconic acid by Pseudomonas putida was studied. The activities of glucose and gluconate dehydrogenases that are responsible for direct oxidation of glucose to 2-ketogluconic acid, were 2-3 times higher during the active growth of the culture than in the stationary phase. The activities of 2-ketogluconokinase and 2-keto-6-phosphogluconate reductase, enzymes converting 2-ketogluconic acid, increased 2-4-fold on the glucose exhausting. The latter enzymes were not active when the culture was grown on nitrogen or phosphorus deficient media, and in this case 2-ketogluconic acid was accumulated in the medium.     

Changes in Activities of Enzymes Involved in General Phenylpropanoid Metabolism during the Induction and Reduction of Anthocyanin Synthesis in a Carrot Suspension Culture as Regulated by 2,4-D

The activities of enzymes involved in general phenylpropanoidmetabolism were followed in a carrot suspension culture duringthe induction and reduction of anthocyanin synthesis regulatedby 2,4-D. When no anthocyanin synthesis occurred in a mediumcontaining 2,4-D (+2,4-D medium), the activities of phenylalanineammonia-lyase (PAL) and 4-coumarate:CoA ligase (4CL) increased1 day after transfer due to the transfer effect, but subsequentlydecreased and remained at a low level. Cinnamate-4-hydroxylase(C4H) activity showed a low level throughout culture. When cellswere transferred to a medium lacking 2,4-D (–2,4-D medium),the activities of PAL, C4H and 4CL increased and maximum activitiesof these enzymes were observed 6–7 days after transfer,when anthocyanin was most rapidly synthesized. When cells were cultured in the –2,4-D medium, the additionof 2,4-D immediately reduced the induced activity of PAL. PALactivity was super-induced by the transfer effect, while anthocyaninsynthesis decreased. The addition of intermediates of generalphenylpropanoid metabolism, with 2,4-D, to the medium 6 daysafter transfer to the –2,4-D medium did not promote anthocyaninsynthesis, whereas dihydroquercetin did promote it. Regulationof anthocyanin synthesis by 2,4-D is discussed in relation tochanges in enzyme activities involved in general phenylpropanoidmetabolism. ¹ Present address: Cell Science and Technology Division, FermentationResearch Institute, Agency of Industrial Science and Technology,Yatabe-machi, Ibaraki 305, Japan. ² Present address: Biological Institute, Faculty of Science,Tohoku University, Sendai 980, Japan.     

Copper extrusion after accumulation during growth of copper-tolerant yeast Yarrowia lipolytica

The Cu2+-tolerant yeast Yarrowia lipolytica accumulated Cu2+ until the late logarithmic phase. Thereafter, Cu2+ was temperature-dependently extruded into phosphate-limited culture medium containing high concentrations of heavy metal ions but not into 10 mM 2-(N-morpholino)ethane sulfonic acid (MES) buffer (pH 6.0). Peptone in the culture medium played an important role in the extrusion, which proceeded even when peptone was substituted with cysteine or histidine, but not with any other amino acid tested.     

Metabolism of n -9, n -6 and n -3 fatty acids in hepatoma Morris 7777 cells. Preferential accumulation of linoleic acid in cardiolipin

The objective of this study was to investigate, using a pulse-chase technique, the different incorporation of (1-(14)C) n -9, n -6 and n 3 fatty acids into hepatoma lipids and their secretion to the culture medium. Docosahexaenoic acid (DHA) accumulated preferentially into the triacylglycerol while arachidonic acid (AA) did into the phospholipid fraction. DHA was poorly secreted to the culture medium whereas AA was secreted to a large extent. The fatty acids were initially esterified mainly into phosphatidylcholine and phosphatidylethanolamine. During the 24 h chase, a general shift from phosphatidylcholine to phosphatidylethanolamine was observed. Linoleic acid was esterified in cardiolipin to a much greater extent than any other fatty acid and it was not converted to more polyunsaturated fatty acids.The supplementation of the culture medium with polyunsaturated fatty acids had no inhibitory effect on the growth of the hepatoma cells, in marked contrast to observations made in other tumoral cells. The reasons for the resistance of the hepatoma cells to polyunsaturated fatty acid toxicity, including the possible antioxidant effect of linoleic acid accumulation in cardiolipin, are also discussed.