Metabolic engineering of Escherichia coli for biosynthesis of d-galactonate

d-galactose is an attractive substrate for bioconversion. Herein, Escherichia coli was metabolically engineered to convert d-galactose into d-galactonate, a valuable compound in the polymer and cosmetic industries. d-galactonate productions by engineered E. coli strains were observed in shake flask cultivations containing 2 g L^?1 d-galactose. Engineered E. coli expressing gld coding for galactose dehydrogenase from Pseudomonas syringae was able to produce 0.17 g L^?1 d-galactonate. Inherent metabolic pathways for assimilating both d-galactose and d-galactonate were blocked to enhance the production of d-galactonate. This approach finally led to a 7.3-fold increase with d-galactonate concentration of 1.24 g L^?1 and yield of 62.0 %. Batch fermentation in 20 g L^?1 d-galactose of E. coli ?galK?dgoK mutant expressing the gld resulted in 17.6 g L^?1 of d-galactonate accumulation and highest yield of 88.1 %. Metabolic engineering strategy developed in this study could be useful for industrial production of d-galactonate.     

Biosynthesis of ethylene glycol in Escherichia coli

Ethylene glycol (EG) is an important platform chemical with steadily expanding global demand. Its commercial production is currently limited to fossil resources; no biosynthesis route has been delineated. Herein, a biosynthesis route for EG production from d-xylose is reported. This route consists of four steps: d-xylose?→?d-xylonate?→?2-dehydro-3-deoxy-d-pentonate?→?glycoaldehyde?→?EG. Respective enzymes, d-xylose dehydrogenase, d-xylonate dehydratase, 2-dehydro-3-deoxy-d-pentonate aldolase, and glycoaldehyde reductase, were assembled. The route was implemented in a metabolically engineered Escherichia coli, in which the d-xylose?→?d-xylulose reaction was prevented by disrupting the d-xylose isomerase gene. The most efficient construct produced 11.7 g?L^?1 of EG from 40.0 g?L^?1 of d-xylose. Glycolate is a carbon-competing by-product during EG production in E. coli; blockage of glycoaldehyde?→?glycolate reaction was also performed by disrupting the gene encoding aldehyde dehydrogenase, but from this approach, EG productivity was not improved but rather led to d-xylonate accumulation. To channel more carbon flux towards EG than the glycolate pathway, further systematic metabolic engineering and fermentation optimization studies are still required to improve EG productivity.     

A revision ofTaraxacum sect.Piesis (Compositae)

On the basis of allozyme and cultivation data, and of additional herbarium material, a taxonomic and nomenclatural revision ofTaraxacum sect.Piesis A.J. Richards exKirschner et?těpánek is provided. The section is made up of halophilous, sexually reproducing taxa. InT. stenocephalum Boiss. etKotschy,T. pindicum Kirschner et?těpánek, sp. nov., andT. perenne Kirschner et?těpánek, sp. nov., a tetraploid chromosome number has been recorded, representing the only known case of sexuality at the tetraploid level in the genus. The complex ofT. stenocephalum, includes some geographically and morphologically extreme populations treated as subspecies: subsp.gumusanicum (Soest)Kirschner et?těpánek, comb. nov., subsp.magnum Kirschner et?těpánek, subsp. nov., and subsp.daralagesicum (Schischk.)Kirschner et?těpánek, comb. nov. In addition toT. bessarabicum (Hornem.)Hand.-Mazz., a widely distributed Eurasian species,T. stenocephalum, a complex centred in Transcaucasia and Anatolia, andT. pachypodum H. Lindb., a North African endemic, four new species are described:T. salsum Kirschner et?těpánek, sp. nov., a diploid endemic confined to E Crimea,T. perenne Kirschner et?těpánek, sp. nov., a tetraploid sexual species known only from SW Crimea,T. pindicum Kirschner et?těpánek, sp. nov., a remarkable tetraploid endemic to the Pindos Mts., Greece, andT. salsitatis Kirschner, ?těpänek etYirdirimli, sp. nov., an Anatolian diploid species. Furthermore, a hybrid betweenT. salsum andT. bessarabicum from Crimea (documented on the basis of allozyme data elsewhere) is given a binomial,T. xmesohalobium Kirschner et?těpánek, nothosp. nov.     

The trilobiteProtostygina and the composition of the Styginidae, with two new genera

The Styginidae is regarded as an exclusively Ordovician family of trilobites, separate from the Scutelluidae. The hitherto poorly known genusProtostygina Prantl &P?ibyl, 1949 is revised. It is recorded with certainty only from the Llanvirn of the Czech Republic, and the type species is a senior synonym of“Raymondaspis” rubensi rubensi P?ibyl &VANěK, 1968 and“R.” rubensi lybar ?najdr, 1976. Two new styginid genera are proposed:Cyrtocybe, with type species“Raymondaspis” turgida Whittington, 1965, is known from the upper Arenig and lower Llanvirn of Newfoundland, Maine and Norway; andPromargo, with type speciesP.forteyi n. sp., occurs in the Arenig of Newfoundland and Spitsbergen.Turgicephalus Fortey, 1980 is regarded as a junior synonym ofRaymondaspis P?ibyl inPrantl &P?ibyl, 1949. Three genera are excluded from the Styginidae:Kirkdomina Tripp, 1962,Pseudostygina Zhou inZhou et al., 1982 andStyginella P?ibyl &Vaněk, 1971.     

d-Tagatose production in the presence of borate by resting Lactococcus lactis cells harboring Bifidobacterium longum l-arabinose isomerase

Bifidobacterium longum NRRL B-41409 l-arabinose isomerase (l-AI) was overexpressed in Lactococcus lactis using a phosphate depletion inducible expression system. The resting L. lactis cells harboring the B. longum l-AI were used for production of d-tagatose from d-galactose in the presence of borate buffer. Multivariable analysis suggested that high pH, temperature and borate concentration favoured the conversion of d-galactose to d-tagatose. Almost quantitative conversion (92 %) was achieved at 20 g L^?1 substrate and at 37.5 °C after 5 days. The d-tagatose production rate of 185 g L^?1 day^?1 was obtained at 300 g L^?1 galactose, at 1.15 M borate, and at 41 °C during 10 days when the production medium was changed every 24 h. There was no significant loss in productivity during ten sequential 24 h batches. The initial d-tagatose production rate was 290 g L^?1 day^?1 under these conditions.     

The effect of some environmental factors on the production of L-DOPA by alginate-entrapped cells of Mucuna pruriens

In-vitro-grown cells of Mucuna pruriens, immobilized in calcium-alginate gels, were able to transform the precursor L-tyrosine into L-dihydroxyphenylalanine (L-DOPA). After the immobilization in alginate the plant cells released 90% of the produced L-DOPA into the medium; supplementation of the medium with calcium inhibited both the transformation of L-tyrosine into L-DOPA and the release of L-DOPA into the medium. Continuous illumination of the beads had a slight beneficial effect on the synthesis of L-DOPA. A simple production medium for the transformation of L-tyrosine into L-DOPA was designed. This medium contained only sucrose and sodium chloride as osmotic stabilizers, a low concentration of calcium chloride for stabilization of the alginate beads, and L-tyrosine as the precursor.     

Observation of algal colonization on Acropora aspera killed by Acanthaster planci

Algae started colonizing branches of the coral Acropora aspera (Dana) killed by the sea star Acanthaster planci (Linnaeus) within less than 24 hours. Two blue-greens ((Microcoleus lyngbyaceus (Crouan) Ag. and Hormothamnion solutum B. & F.)) dominated the early community but became less abundant than a brown ((Giffordia indica (Sonder) Papenfuss & Chihara)) after 26 days.     

Engineered Bacillus subtilis 168 produces l-malate by heterologous biosynthesis pathway construction and lactate dehydrogenase deletion

In the present work, Bacillus subtilis was engineered to produce l-malate. Initially, the study revealed that the slight fumarase activity under anaerobic conditions is extremely favourable for l-malate one-step fermentation accumulation. Subsequently, an efficient heterologous biosynthesis pathway formed by Escherichia coli phosphoenolpyruvate carboxylase and Saccharomyces cerevisiae malate dehydrogenase was introduced into B. subtilis, which led to 6.04?±?0.19?mM l-malate production. Finally, the l-malate production was increased 1.5-fold to 9.18?±?0.22?mM by the deletion of lactate dehydrogenase. Under two-stage fermentation conditions, the engineered B. subtilis produced up to 15.65?±?0.13?mM l-malate, which was 86.3?% higher than that under anaerobic fermentation conditions. Though the l-malate production by the recombinant was low, this is the first attempt to produce l-malate in engineered B. subtilis and paves the way for further improving l-malate production in B. subtilis.     

The Effects of Magnesium, l-Carnitine, and Concurrent Magnesium–l-Carnitine Supplementation in Migraine Prophylaxis

Given the conflicting results about the positive effects of magnesium and l-carnitine and as there is no report concerning concurrent supplementation of magnesium and l-carnitine on migraine prophylaxis, the effects of magnesium, l-carnitine, and concurrent magnesium?Cl-carnitine supplementation on migraine indicators were assessed. In this clinical trial, 133 migrainous patients were randomly assigned into three intervention groups: magnesium oxide (500?mg/day), l-carnitine (500?mg/day), and Mg?Cl-carnitine (500?mg/day magnesium and 500?mg/day?l-carnitine), and a control group. After 12?weeks of supplementation, the checklist of migraine indicators including migraine attacks/month, migraine days/month, and headache severity was completed, and serum concentrations of magnesium and l-carnitine were measured by atomic absorption spectrophotometry and enzymatic UV test, respectively. The results showed a significant reduction in all migraine indicators in all studied groups (p?<?0.05). The ANOVA results showed a significant reduction in migraine frequency across various supplemented and control groups (p?=?0.008). By separating the effects of magnesium supplementation from other confounding factors such as routine treatments using the repeated measures and nested model, it was clarified that magnesium supplementation had a significant effect on all migraine indicators. Oral supplementation with magnesium oxide and l-carnitine and concurrent supplementation of Mg?Cl-carnitine besides routine treatments could be effective in migraine prophylaxis; however, larger trials are needed to confirm these preliminary findings.     

Improvement of l-lactic acid production by osmotic-tolerant mutant of Lactobacillus casei at high temperature

l-Lactic acid production by Lactobacillus casei was used as a model to study the mechanism of substrate inhibition and the strategy for enhancing l-lactic acid production. It was found that the concentration of cell growth and l-lactate decreased with the increase of glucose concentration and fermentation temperature. To enhance the osmotic stress resistance of the strain at high temperature, a mutant G-03 was screened and selected with 360?g/L glucose at 45°C as the selective criterion. To further increase the cell growth for lactic acid production, 3?g/L of biotin was supplemented to the medium. As a result, l-lactate concentration by the mutant G-03 reached 198.2?g/L (productivity of 5.5?g?L^?1?h^?1) at 41°C in a 7-L fermentor with 210?g/L glucose as carbon source. l-Lactate concentration and productivity of mutant G-03 were 115.2% and 97.8% higher than those of the parent strain, respectively. The strategy for enhancing l-lactic acid production by increasing osmotic stress resistance at high temperature may provide an alternative approach to enhance organic acid production with other strains.     

Effects of Dietary Zinc and l-Arginine Supplementation on Total Antioxidants Capacity, Lipid Peroxidation, Nitric Oxide, Egg Weight, and Blood Biochemical Values in Japanase Quails

The aim of this study was to evaluate effects of dietary zinc and l-arginine supplementation on blood total antioxidant capacity (TAC), malondialdehyde (MDA), nitric oxide (NO), some blood chemistry parameters, and egg weights of laying quails. Three groups of Japanese quails were fed with a diet containing l-arginine (5 mg/kg), zinc (60 mg/kg), and normal basal diet (control) for 30 days. TAC, lipid peroxidation, and biochemical analysis were performed in the blood of animals. l-Arginine and zinc supplementation improved TAC and reduced MDA concentrations compared to the control (P?<?0.05). In comparison to the control, blood NO concentrations were increased by l-arginine (P?<?0.01) and zinc treatment (P?<?0.05). Both zinc (P?<?0.001) and l-arginine (P?<?0.01) supplementation significantly increased egg weight in laying quails. Some of the blood chemistry parameters were also altered by the treatment of l-arginine and zinc supplementation. No difference was found in blood albumin and creatinine levels among the groups. Blood glucose (P?=?0.833) and total protein (P?=?0.264) levels in control and l-arginine-treated groups were found to be similar. Glucose and total protein levels were decreased in zinc-supplemented animals compared to the control and l-arginine groups (P?<?0.05). No difference was found in triglyceride levels between control and zinc-applied groups (P?=?0.197). However, l-arginine treatment reduced the blood triglyceride levels compared to the control (P?<?0.05). In conclusion, l-arginine and zinc supplementation could be beneficial and effective for decreasing oxidative stress, boosting antioxidant capacity, and improving egg weight in the blood of the animals.     

Karyological and taxonomic observations onDracocephalum foetidum Bunge andKoenigia islandica L.

Chromosome numbers are reported for two Mongolian species,Dracocephalum foetidum Bunge (2n=12) andKoenigia islandica L. (2n=14). The relationship ofD. foetidum toD. moldavica L. (2n=10) and some patterns of phenotypic variation inK. islandica are briefly discussed. The following new combinations are proposed:K. cyanadra (Diels) Měsí?ek etSoják,K. forrestii (Diels) Měsí?ek etSoják,K. hubertii (Lingelsh.) Měsí?ek etSoják, andK. nummularifolia (Meisn.) Měsí?ek etSoják.     

A new savanna-like community of the sierra del rosario mountains,Cuba

This communication describes a new savanna-like community, theBletio purpureae-Andropogonetum gracilis Balátová-Tulá?ková etR. Capote ass. nov., found in the woodless eastern part of the Sierra del Rosario Mountains (western part of Cuba). From the phytosociological point of view, it belongs to the allianceAchlaenion piptostachyae Balátová-Tulá?ková all. nov., the orderAchlaenetalia piptostachyae Balátová-Tulá?ková ord. nov., and the classSclerio baldwinii-Andropogonetea gracilis Balátová-Tulá?ková cl. nov. In the area under study four subassociations were distinguished: theBletio-Andropogonetum typicum Balátová-Tulá?ková et.R. Capote subass. nov., theB.-A. rhynchosporetosum fascicularis Balátová-Tulá?ková etR. Capote subass. nov., theB.-A. cassietosum aeschynomenes Balátová-Tulá?ková etR. Capote subass. nov., and theB.-A. stenandrietosum droseroidis Balátová-Tulá?ková etR. Capote subass. nov.     

Erwägungen über die Taxonomie und Nomenklatur der GattungActinotaenium Teil

In der botanischen Literatur werden oft neue Kombinationen und neue Namen ungültig publiziert, weil die Vorschriften des Art. 33 d. Int. Codes nicht eingehalten werden. Die Verfasser verfolgen diese Frage bei der GattungActinotaenium Teiling 1954 (Fam.Desmidiaceae, KlasseConjugatophyceae der Grünalgen), weisen auf die Unklarheiten und Unrichtigkeiton in ihrer Systematik hin und sind bestrebt, diese zu verbessern, soweit es bei heutigem Zustand der Kenntnisse möglich ist. Im Artikel ist ein neues TaxonActinotaenium messikommeri R??i?ka etPouzar beschrieben und folgende neue Kombinationen veröffentlicht;Actinotaenium adelochondrum var.kriegeri (Messik.) R??i?ka, A.angulatum (W. etG. S. West)R??i?ka etPouzar,A. angulatum var.brasiliense (Grönblad) R??i?ka etPouzar,A. cordanum (Bréb.) R??i?ka etPouzar,A. gelidum (Wittr.) R??i?ka,A. heterotaphridium (W. etG. S. West)R??i?ka etPouzar undA. hibernicum (W. West) R??i?ka.     

DieFestucetalia valesiacae-Gesellschaften m Ostteil des Gebirges České středohoří (Böhmisches Mittelgebirge) 2. Synökologie,Sukzession und syntaxonomische Ergänzungen

Die vorliegende Arbeit befasst sich mit zwei, die Vegetation felsiger vulkanischer Substrate einschliessenden Verbänden:Alysso-Festucion pallentis Moravec 1967 undFestucion valesiacae Klika 1933, in deren Rahmen acht Assoziationen untersucht wurden:Alysso-Festucetum Klika ex?e?ovský 1949,Asperulo-Festucetum Preis inKlika 1939,Allio-Sedetum Klika 1939,Melico-Sempervivetum Preis inKlika 1939,Alysso-Potentilletum Preis 1939,Carici-Festucetum Klika 1951,Erysimo-Festucetum Klika 1933 undFestuco-Stipetum Sillinger 1931. Im ökologischen Abschnitt wurde auf den Vergleich von chemischen eigenschaften der Böden (Azidität und ihr Saisonverlauf, Gehalt an Austauschionen—H⁺, Al³⁺, Ca²⁺, Mg²⁺ und an C und N) und deren morphologischen Eigenschaften (Tiefe und Stratigraphie der Bodenhorizonte, ihre Beschreibung) Nachdruck gelegt. Ebenso wurde dem Standort und den Beziehungen der Gesellschaft zu den anschliessend benachbarten Gesellschaften an der Lokalität Aufmerksamkeit gewidmet. In der Arbeit werden auch einige selten vorkommende oder noch nicht beschriebene Syntaxa erwähnt, die mit den oben erwähnten Assoziationen in Kontakt stehen:Calluno vulgaris-Cytisetum nigricantis Prsg. 1953 em.Oberd. 1957,Polytricho piliferi-Scleranthetum perennis Moravec 1967,Geranio sanguinei-Dictamnetum albae Wendelberger 1954,Geranio sanguinei-Trifolietum alpestris Th. Müller 1961,Dictamno albae-Sorbetum Knapp 1942; weiter die neubeschriebenen Syntaxa:Alysso saxatilis-Festucetum duriusculae cotoneasteretosum integerrimae, Potentillo opacae-Festucetum sulcatae pulsatilletosum patentis mit Var.Vaccinium myrtillus und Var.Pimpinella saxifraga und Subass.eryngietosum campestris, Pulsatillo pratensis-Avenochloetum pratensis, Koelerio macranthae-Stipetum joannis mit fünf Subassoziationen:verbascetosum lychnitis, stipetosum pulcherrimae, stipetosum smirnovii, stipetosum glabratae undstipetosum tirsae undErysimo crepidifolii-Festucetum valesiacae dictamnetosum albae. Alle Syntaxa sind mit Aufnahmematerial belegt und ihre Sukzessions- und topographischen Beziehungen werden beschrieben.     

Holocene anthropogenic landscapes in the Balkans: the palaeobotanical evidence from southwestern Bulgaria

Palaeoecological reconstructions from the region of southwestern Bulgaria were used for inferring the human impact on the vegetation and landscape during the last 8 millennia. They are based on data from pollen analyses of lakes and peat-bogs, plant macrofossils, archaeobotanical finds and radiocarbon dating. During the early Holocene, after 7900?cal. b.p. (5950?cal. b.c.) the climate changed to cooler summers, milder winters and higher precipitation resulting in the formation of a coniferous belt dominated by Pinus sp. and Abies alba. These favorable environmental pre-conditions had a positive influence on the Neolithisation of the Balkans after the 8200?cal. b.p. (6250?cal. b.c.) cold event, which caused drought in the Eastern Mediterranean. Direct evidence from wood charcoal records from the Neolithic settlement layers in the study area shows a slight modification of the surrounding woodlands and an increase of the light-demanding components, probably expressed through larger forest border zones and thinning out of the wood stands. The increase in the number of settlements in the valleys of southwestern Bulgaria intensified the human activity visible in the palaeobotanical record from 6950?cal. b.p. (5000?cal. b.c.) onwards. Between ca. 5700–5100?cal. b.p. (3800–3200?cal. b.c.) signs of anthropogenic influence on the vegetation are virtually absent. The intensity of human impact increased notably after 3200?cal. b.p. (1400–1250?cal. b.c., approx. Late Bronze Age), documented by a rise of pollen anthropogenic indicators. The final transformations in the natural forest cover after 2750?cal. b.p. (800?cal. b.c. onset of the Iron Age) marked the reduction of the coniferous forests dominated by Abies alba and Pinus sp. and the expansion of Fagus sylvatica and Picea abies. These vegetation changes are contemporaneous with increase of the palaeofire activities and the next peak of anthropogenic indicators. The changes in the landscape during the Roman period and the medieval period reflect regional environmental features and were forced by the diversification of anthropogenic activity.     

Characterization of uptake and release processes ford- andl-aspartate in primary cultures of astrocytes and cerebellar granule cells

The uptake ofl-andd-aspartate was studied in astrocytes cultured from prefrontal cortex and in granule cells cultured from cerebellum. A high affinity uptake system forl- andd-aspartate was found in both cell types, and the two stereoisomers exhibited essentially the sameK _m - andV _max -values in bouth astrocytes (l-aspartate:K _m 77 μM;V _max 11.8 nmol×min^?1×mg^?1;d-aspartate:K _m 83 μM;V _max 14.0 nmol×min^?1×mg^?1) and granule cells (l-aspartate:K _m 32 μM;V _max 2.8 nmol ×min^?1×mg^?1;d-aspartate:K _m 26 μM;V _max 3.0 nmol×min^?1×mg^?1). To investigate whetherl-glutamate,l-aspartate andd-aspartate use the same uptake system a detailed kenetic analysis was performed. The uptake kinetics of each one of the three amino acids was studied in the presence of the two other amino acids, and no essential differences between the uptake characteristics of the amino acids were found. In addition to the uptake studies the release ofD-aspartate from cerebellar granule cells was investigated and compared withl-glutamate release. A Ca²⁺-dependent, K⁺-induced release was found for both amino acids.     

The human L-pipecolic acid oxidase is similar to bacterial monomeric sarcosine oxidases rather than D-amino acid oxidases

L-Pipecolic acid oxidase activity is deficient in patients with peroxisome biogenesis disorders (PBDs). Because its role, if any, in these disorders is unknown, the authors cloned the human gene to order to further study its functions. BLAST search of the translated sequence showed greatest homology to Bacillus sp. NS-129 monomeric sarcosine oxidase. The purified enzyme could use either L-pipecolic acid or sarcosine as a substrate. No homology was found to the peroxisomal D-amino acid oxidases. A further comparison of L-pipecolic acid oxidase to the two D-amino acid oxidases in peroxisomes showed that the proteins differed in many ways. First, both D-amino acid oxidase and L-pipecolic acid oxidase showed no enzyme activity in liver from Zell-weger syndrome patients; D-aspartate oxidase activity was unchanged from control levels. Although all were targeted to peroxisomes, their targeting signals differed. No L-pipecolic acid oxidase was found in brain or other tissues outside of liver and kidney. The D-amino acid oxidases were similarly and more widely distributed. Finally, although D-amino acid degradation is limited to peroxisomes in mammals, L-pipecolic acid can be oxidized in either mitochondria or peroxisomes, or both.     

Engineering lower inhibitor affinities in β-d-xylosidase

β-d-Xylosidase catalyzes hydrolysis of xylooligosaccharides to d-xylose residues. The enzyme, SXA from Selenomonas ruminantium, is the most active catalyst known for the reaction; however, its activity is inhibited by d-xylose and d-glucose (K _i values of ～10^?2?M). Higher K _i’s could enhance enzyme performance in lignocellulose saccharification processes for bioethanol production. We report here the development of a two-tier high-throughput screen where the 1° screen selects for activity (active/inactive screen) and the 2° screen selects for a higher K _i(d-xylose) and its subsequent use in screening ～5,900 members of an SXA enzyme library prepared using error-prone PCR. In one variant, termed SXA-C3, K _i(d-xylose) is threefold and K _i(d-glucose) is twofold that of wild-type SXA. C3 contains four amino acid mutations, and one of these, W145G, is responsible for most of the lost affinity for the monosaccharides. Experiments that probe the active site with ligands that bind only to subsite ?1 or subsite +1 indicate that the changed affinity stems from changed affinity for d-xylose in subsite +1 and not in subsite ?1 of the two-subsite active site. Trp145 is 6 Å from the active site, and its side chain contacts three active-site residues, two in subsite +1 and one in subsite ?1.     

Bemerkungen zur Variabilität vonSaxifraga marginata

Saxifraga marginata Sternb. is divided into three subspecies:S. m. subsp.marginata, S. m. subsp.bubakii (Rohlena)Chrtek etSoják,S. m. subsp.karadzicensis (Degen etKo?anin)Chrtek etSoják (environments of Skopje, Macedonia).