Performance analyses of a pH-shift and DOT-shift integrated fed-batch fermentation process for the production of ganoderic acid and Ganoderma polysaccharides by medicinal mushroom Ganoderma lucidum

Investigations on Ganoderma lucidum fermentation suggested that the responses of the cell growth and metabolites biosynthesis to pH and dissolved oxygen tension (DOT) were different. The ganoderic acid (GA) production of 321.6 mg/L was obtained in the pH-shift culture by combining a 4-day culture at pH 3.0 with the following 6-day culture at pH 4.5, which was higher by 45% and 300% compared with the culture at pH 3.0 and 4.5, respectively. The GA production of 487.1 mg/L was achieved in the DOT-shift culture by combining a 6-day culture at 25% of DOT with a following 6-day culture at 10% of DOT, which was higher by 43% and 230% compared with the culture at 25% and 10% of DOT, respectively. A fed-batch fermentation process by combining the above-mentioned pH-shift and DOT-shift strategies resulted in a significant synergistic enhancement of GA accumulation up to 754.6 mg/L, which is the highest reported in the submerged fermentation of G. lucidum in stirred-tank bioreactor.     

ATP production after pH shift in membrane vesicles from maize coleoptiles

Microsomal membrane vesicles and purified plasma membranevesicles obtained from coleoptiles of maize (Zea mays L.) weresubjected to pH shifts from pH 7.8 to 4.7. In the presence of ATPaseinhibitors such as vanadate, net accumulation of radiolabelled butyricand indole-3-acetic acid (IAA) remained higher than in controls. When 2min after the pH shift, at 4°C, the microsomal vesicles weredenatured the amount of ATP could be determined using theluciferin/luciferase assay. Significantly increased ATP production overcontrol values – no pH-shift or ionophore treatment – wasfound. Therefore, such vesicles might produce ATP for in vitrotransport processes such as auxin efflux.     

Elicitation of rosmarinic acid by <Emphasis Type="Italic">Lavandula vera</Emphasis> MM cell suspension culture with abiotic elicitors

The growth of Lavandula vera MM plant cell suspension culture and rosmarinic acid biosynthesis under elicitation with benzothiadiazole and methyl jasmonate were investigated. Upon elicitation with 50 μM methyl jasmonate, the production of rosmarinic acid was enhanced 2.4-fold (3348 mg/l) compared to the non-elicited cells. The influence of benzothiadiazole on rosmarinic acid biosynthesis was weaker and 12 h after its addition the achieved yields were 20–30% higher compared to the control variant at this time. The influence of both elicitors on rosmarinic acid secretion into the culture medium was also discussed.     

Production of succinate by a <Emphasis Type="Italic">pfl</Emphasis>B <Emphasis Type="Italic">ldh</Emphasis>A double mutant of <Emphasis Type="Italic">Escherichia coli</Emphasis> overexpressing malate dehydrogenase

The gene encoding malate dehydrogenase (MDH) was overexpressed in a pflB ldhA double mutant of Escherichia coli, NZN111, for succinic acid production. With MDH overexpression, NZN111/pTrc99A-mdh restored the ability to metabolize glucose anaerobically and 0.55 g/L of succinic acid was produced from 3 g/L of glucose in shake flask culture. When supplied with 10 g/L of sodium bicarbonate (NaHCO₃), the succinic acid yield of NZN111/pTrc99A-mdh reached 1.14 mol/mol glucose. Supply of NaHCO₃ also improved succinic acid production by the control strain, NZN111/pTrc99A. Measurement of key enzymes activities revealed that phosphoenolpyruvate (PEP) carboxykinase and PEP carboxylase in addition to MDH played important roles. Two-stage culture of NZN111/pTrc99A-mdh was carried out in a 5-L bioreactor and 12.2 g/L of succinic acid were produced from 15.6 g/L of glucose. Fed-batch culture was also performed, and the succinic acid concentration reached 31.9 g/L with a yield of 1.19 mol/mol glucose.     

A novel alkalo- and thermostable phospholipase D from <Emphasis Type="Italic">Streptomyces olivochromogenes</Emphasis>

A 60 kDa phospholipase D (PLD) was obtained from Streptomyces olivochromogenes by one-step chromatography on Sepharose CL-6B. Maximal activity was at pH 8 and 75°C and the enzyme was stable from pH 7 to 13 and from 55 to 75°C. Thermal and pH stability with temperature optimum of the enzyme were highest among Streptomyces PLDs reported so far. The activity was Ca²⁺-dependent and enhanced by detergents. The Km and Vmax values for phosphatidylcholine were 0.6 mM and 650 μmol min⁻¹ mg⁻¹, respectively. In addition, the enzyme also revealed transphosphatidylation activity, which was optimum at pH 8 and 50°C. The first 15 amino acid residues of the N terminal sequence were ADYTPGAPGIGDPYY, which are significantly different from the other known PLDs. The enzyme may therefore be a novel PLD with potential application in the lipid industry.     

Effect of medium-pH and MES on adventitious root formation from stem disks of apple

We have examined the effect of medium-pH on rooting using 1-mm slices cut from stems of apple microshoots. Before autoclaving, the pH of the rooting medium was set at various pH values between 4.5 and 8.0. During autoclaving, the pH drifted in particular in the alkaline region. Additional changes occurred during culture and the range set at 4.5–8.0 had shifted to 5.2–6.0 after autoclaving and 3 weeks of culture. When 10 mM 2-(N-morpholino)ethanesulfonic acid (MES) had been added as buffering agent, the pH was stable when set at 5.0–6.5. Highest rooting was achieved at pH ~5.3 with and without MES (pH measured after autoclaving). This maximum did not correlate with highest auxin uptake. MES inhibited adventitious root formation during the initial phase of root formation when the meristemoids are being formed (ca. 30% reduction at 10 mM) but was promotive during outgrowth of the meristemoids to roots (30% increase at 10 mM). Inhibition and promotion by MES were not related to its buffering action as they were observed at all pHs.     

Production of cyclic adenosine monophosphate by Arthrobacter sp. A302 using fed-batch fermentation with pH-shift control

The production of cyclic adenosine monophosphate (cAMP) by Arthrobacter sp. A302 was studied in a 5 L stirred tank fermentor under a range of pH values (6.5–8.0) and glucose feeding rates. In batch fermentation under a controlled pH, the optimum pH for cell growth was 7.5 with dry cell density (X) of 11.43 g L, and the optimum pH for cAMP accumulation was 7.0 with cAMP concentration of 7.41 g L. In order to achieve the high X and cAMP yield simultaneously, a pH-shift control strategy was proposed based on kinetic analysis of specific cell growth rate (μ) and specific cAMP formation rate (q _s ). In this method, pH was controlled to 7.0 for the first 30 h of fermentation, and then subsequently shifted to 7.5 and maintained until the end of the process. Application of this approach significantly enhanced the cAMP concentration. Thereafter, cAMP production was further improved by combining the above-mentioned pH-control system and fed-batch process with glucose at a constant feeding rate of 1.0 g L⁻¹ h⁻¹. Under optimum conditions, the final cAMP production was 10.87 g L, which is 110.0, 46.7, and 27.7% higher than that of the pH-uncontrolled, pH-controlled, and pH-shift controlled methods, respectively.     

The effect of manipulating ecdysteroid signaling on embryonic eye development in the locust<Emphasis Type="Italic"> Schistocerca americana</Emphasis>

Adult body plan differentiation in holometabolous insects depends on global induction and control by ecdysteroid hormones during the final phase of postembryogenesis. Studies in Drosophila melanogaster and Manduca sexta have shown that this pertains also to the development of the compound eye retina. It is unclear whether the hormonal control of postembryonic eye development in holometabolous insects represents evolutionary novelty or heritage from hemimetabolous insects, which develop compound eyes during embryogenesis. We therefore investigated the effect of manipulating ecdysteroid signaling in cultured embryonic eye primordia of the American desert locust Schistocerca americana, in which ecdysteroid level changes are known to induce three rounds of embryonic molt. Although at a considerably reduced rate compared to in vivo development, early differentiation and terminal maturation of the embryonic retina was observed in culture even if challenged with the ecdysteroid antagonist cucurbitacin B. Supplementing cultures with 20-hydroxyecdysone (20E) accelerated differentiation and maturation, and enhanced cell proliferation. Considering these results, and the relation between retina differentiation and ecdysteroid level changes during locust embryogenesis, we conclude that ecdysteroids are not an essential but possibly a modulatory component of embryonic retina development in S. americana. We furthermore found evidence that 20E initiated precocious epithelial morphogenesis of the posterior retinal margin indicating a more general role of ecdysteroids in insect embryogenesis.Electronic Supplementary Material Supplementary material is available in the online version of this article at Edited by C. Desplan     

A <Emphasis Type="Italic">Sordaria macrospora</Emphasis> mutant lacking the <Emphasis Type="Italic">leu1</Emphasis> gene shows a developmental arrest during fruiting body formation

Developmental mutants with defects in fruiting body formation are excellent resources for the identification of genetic components that control cellular differentiation processes in filamentous fungi. The mutant pro4 of the ascomycete Sordaria macrospora is characterized by a developmental arrest during the sexual life cycle. This mutant generates only pre-fruiting bodies (protoperithecia), and is unable to form ascospores. Besides being sterile, pro4 is auxotrophic for leucine. Ascospore analysis revealed that the two phenotypes are genetically linked. After isolation of the wild-type leu1 gene from S. macrospora, complementation experiments demonstrated that the gene was able to restore both prototrophy and fertility in pro4. To investigate the control of leu1 expression, other genes involved in leucine biosynthesis specifically and in the general control of amino acid biosynthesis (“cross-pathway control”) have been analysed using Northern hybridization and quantitative RT-PCR. These analyses demonstrated that genes of leucine biosynthesis are transcribed at higher levels under conditions of amino acid starvation. In addition, the expression data for the cpc1 and cpc2 genes indicate that cross-pathway control is superimposed on leucine-specific regulation of fruiting body development in the leu1 mutant. This was further substantiated by growth experiments in which the wild-type strain was found to show a sterile phenotype when grown on a medium containing the amino acid analogue 5-methyl-tryptophan. Taken together, these data show that pro4 represents a novel mutant type in S. macrospora, in which amino acid starvation acts as a signal that interrupts the development of the fruiting body. Electronic Supplementary Material Supplementary material is available for this article at http://dx.doi.org/10.1007/s00438-005-0021-8     

High resolution crystal structure of Rubrivivax gelatinosus cytochrome c′

The structure of the cytochrome c′ from the purple non-sulfur phototrophic bacterium Rubrivivax gelatinosus was determined using two crystals grown independently at pH 6.3 and pH 8. The resolution attained for the two structures (1.29 Å and 1.50 Å for the crystals at high and low pH, respectively) is the highest to date for this class of proteins. The two structures were compared in detail in an attempt to investigate the influence of pH on the geometry of the haem and of the coordination environment of the Fe(III) ion. However, while the results suggest some small propensity for the movement of the metal atom out of the plane of the haem ring upon pH increase, the accuracy of the measurements at these two pH below the pK of the axial histidine is not sufficient to provide hard evidence of a shift in the iron position and associated changes.     

Short-term kinetics of elongation growth of gibberellin-responsive lettuce hypocotyl sections

The short-term kinetics of growth of the excised lettuce (Lactuca sativa L.) hypocotyl were characterized with respect to the effects of gibberellic acid (GA₃), indole-3-acetic acid (IAA), KCl and pH. A Hall-device-based, miniaturized, linear displacement transducer was developed to measure the growth of 2-mm hypocotyl sections with 1-m resolution. Following treatment with GA₃, a lag time of less than 10 min was typically followed by an increase in growth rate with two acceleration phases, reaching a final elevated rate within about 1 h. The kinetics of the response to GA₁, a mixture of GA₄ and GA₇, and GA₉ were similar to the response to GA₃. There was no response to IAA treatment either in the presence or absence of GA₃. KCl alone had no effect on the growth rate, but caused an increase in rate when added after GA₃, with a lag time of usually less than 1 h. Responses to pH changes had lag times of a few minutes in all cases. A shift from H₂O to pH 6 buffer inhibited growth, while a shift from H₂O to pH 4 buffer resulted in a transient increase to a rate comparable to that induced by GA₃. A shift from pH 6 to pH 5 caused an increase in growth rate, followed by a gradual decline to an H₂O control rate after more than an hour. The responses to GA₃ at pH 4 and pH 5 were similar to that found for addition of GA₃ to water controls.Abbreviations GA gibberellin - GA₃ gibberellic acid - GA₁, GA₄₊₇, GA₉ gibberellins A₁, A₄₊₇, A₉ - IAA indole-3-acetic acid     

Gliclazide Microcrystals Prepared by Two Methods of <Emphasis Type="Italic">In Situ</Emphasis> Micronization: Pharmacokinetic Studies in Diabetic and Normal Rats

The low water-solubility of gliclazide (GL) leads to a low dissolution rate and variable bioavailability. The aim of this study was to investigate the effect of micronization on the absorption and pharmacokinetics of GL after oral administration in rats. GL microcrystals were prepared using solvent-change and pH-shift methods. Scanning electron microscopy showed considerable changes in the shape and size of crystals using both methods. In the optimized formulation of each method, the particle size of treated GL was reduced about 30 (from 290 to 9.9 μm) and 61 times (to 4.76 μm) by solvent-change and pH-shift methods, respectively. Recrystallized samples showed faster dissolution rate than untreated GL particles. Glucose-lowering effect, C _max, and area under the drug concentration-time profile (area under the curve (AUC)) were compared in diabetic and normal rats. AUC and C _max were increased by microcrystals in both groups of animals. Administration of 40 mg/kg of GL in the form of untreated drug and microcrystals obtained by solvent-change and pH-shift methods caused 12.49% and 21.04% enhancement in glucose-lowering effect of GL in diabetic rats, respectively.     

The adaptive acid tolerance response in root nodule bacteria and Escherichia coli

Root nodule bacteria and Escherichia coli show an adaptive acid tolerance response when grown under mildly acidic conditions. This is defined in terms of the rate of cell death upon exposure to acid shock at pH 3.0 and expressed in terms of a decimal reduction time, D. The D values varied with the strain and the pH of the culture medium. Early exponential phase cells of three strains of Rhizobium leguminosarum (WU95, 3001 and WSM710) had D values of 1, 6 and 5 min respectively when grown at pH 7.0; and D values of 5, 20 and 12 min respectively when grown at pH 5.0. Exponential phase cells of Rhizobium tropici UMR1899, Bradyrhizobium japonicum USDA110 and peanut Bradyrhizobium sp. NC92 were more tolerant with D values of 31, 35 and 42 min when grown at pH 7.0; and 56, 86 and 68 min when grown at pH 5.0. Cells of E. coli UB1301 in early exponential phase at pH 7.0 had a D value of 16 min, whereas at pH 5.0 it was 76 min. Stationary phase cells of R. leguminosarum and E. coli were more tolerant (D values usually 2 to 5-fold higher) than those in exponential phase. Cells of R. leguminosarum bv. trifolii 3001 or E. coli UB1301 transferred from cultures at pH. 7.0 to medium at pH 5.0 grew immediately and induced the acid tolerance response within one generation. This was prevented by the addition of chloramphenicol. Acidadapted cells of Rhizobium leguminosarum bv. trifolii WU95 and 3001; or E. coli UB1301, M3503 and M3504 were as sensitive to UV light as those grown at neutral pH.