<Emphasis Type="Italic">In vitro</Emphasis> Blastogenesis inhibited by <Emphasis Type="Italic">Erwinia carotovora</Emphasis><Emphasis Type="SmallCaps">L</Emphasis>-Asparaginase

Escherichia coliL-asparaginase, an antileukaemic agent in man¹, inhibits in vitro mitogen or antigen-induced blastogenesis in man^2,3 and in animals (M. Bennett, E. G. Mayhew and T. Han, unpublished data) and suppresses bone-marrow derived antibody precursor cells in the mouse⁴. We now report that another L-asparaginase preparation—from Erwinia carotovora—also possesses antileukaemic activity^5,6 and has a more pronounced immunosuppressive effect on in vitro blastogenesis than the E. coli enzyme.     

Inactivation of Complement by <Emphasis Type="SmallCaps">L</Emphasis>-Asparaginase Preparations not Correlated with Enzyme Content

DELÁGE et al.¹ recently reported that L-asparaginase (L-A) in vitro inhibits human and guinea-pig whole serum complement and that L-A is a potent activator of C1. They therefore proposed that their results may “give a clue to the severe anaphylactic reactions noted in some patients on administration of the drug”. Because guinea-pig serum contains L-A in high quantities and Deláge et al. used reagents and methods that make their data difficult to interpret on a molecular basis, we have reinvestigated the effect of L-A preparations on human and guinea-pig complement.     

Morphological and molecular characterization of cyanobacteria from a Brazilian facultative wastewater stabilization pond and evaluation of microcystin production

The cyanobacterial population in the Cajati waste stabilization pond system (WSP) from São Paulo State, Brazil was assessed by cell isolation and direct microscope counting techniques. Ten strains, belonging to five genera (Synechococcus, Merismopedia, Leptolyngbya, Limnothrix, and Nostoc), were isolated and identified by morphological and molecular analyses. Morphological identification of the isolated strains was congruent with their phylogenetic analyses based on 16S rDNA gene sequences. Six cyanobacterial genera (Synechocystis, Aphanocapsa, Merismopedia, Lyngbya, Phormidium, and Pseudanabaena) were identified by direct microscope inspection. Both techniques were complementary, since, of the six genera identified by direct microscopic inspection, only Merismopedia was isolated, and the four other isolated genera were not detected by direct inspection. Direct microscope counting of preserved cells showed that cyanobacteria were the dominant members (>90%) of the phytoplankton community during both periods evaluated (summer and autumn). ELISA tests specific for hepatotoxic microcystins gave positive results for six strains (Synechococcus CENA108, Merismopedia CENA106, Leptolyngbya CENA103, Leptolyngbya CENA112, Limnothrix CENA109, and Limnothrix CENA110), and for wastewater samples collected from raw influent (3.70 μg microcystins/l) and treated effluent (3.74 μg microcystins/l) in summer. Our findings indicate that toxic cyanobacteria in WSP systems are of concern, since the treated effluent containing cyanotoxins will be discharged into rivers, irrigation channels, estuaries, or reservoirs, and can affect human and animal health.     

Crystal Structure of the Protein <Emphasis Type="SmallCaps">l</Emphasis>-Isoaspartyl Methyltransferase from <Emphasis Type="Italic">Escherichia coli</Emphasis>

Among the known covalent damages that can occur spontaneously to proteins, the formation of isoaspartyl linkages through deamidation of asparagines and isomerization of aspartates may be one of the most rapid forms under conditions of physiological pH and temperature. The protein l-isoaspartyl methyltransferase (PIMT) is thought to recognize l-isoaspartyl residues and repair this kind of damaged proteins. Curiously, there is a potential functional difference between bacterial and mammalian PIMTs. Herein, we present the crystal structure of Escherichia coli PIMT (EcPIMT) at a resolution of 1.8 Å. The enzyme we investigated was able to remain bound to its product S-adenosylhomocysteine (SAH) during crystallization. Analysis indicates that the high affinity of EcPIMT for SAH might lead to the lower activity of the enzyme.     

Phosphoenolpyruvate:glucose phosphotransferase system modification increases the conversion rate during <Emphasis Type="SmallCaps">l</Emphasis>-tryptophan production in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Escherichia coli FB-04(pta1), a recombinant l-tryptophan production strain, was constructed in our laboratory. However, the conversion rate (l-tryptophan yield per glucose) of this strain is somewhat low. In this study, additional genes have been deleted in an effort to increase the conversion rate of E. coli FB-04(pta1). Initially, the pykF gene, which encodes pyruvate kinase I (PYKI), was inactivated to increase the accumulation of phosphoenolpyruvate, a key l-tryptophan precursor. The resulting strain, E. coli FB-04(pta1)ΔpykF, showed a slightly higher l-tryptophan yield and a higher conversion rate in fermentation processes. To further improve the conversion rate, the phosphoenolpyruvate:glucose phosphotransferase system (PTS) was disrupted by deleting the ptsH gene, which encodes the phosphocarrier protein (HPr). The levels of biomass, l-tryptophan yield, and conversion rate of this strain, E. coli FB-04(pta1)ΔpykF/ptsH, were especially low during fed-batch fermentation process, even though it achieved a significant increase in conversion rate during shake-flask fermentation. To resolve this issue, four HPr mutations (N12S, N12A, S46A, and S46N) were introduced into the genomic background of E. coli FB-04(pta1)ΔpykF/ptsH, respectively. Among them, the strain harboring the N12S mutation (E. coli FB-04(pta1)ΔpykF-ptsHN12S) showed a prominently increased conversion rate of 0.178 g g^?1 during fed-batch fermentation; an increase of 38.0% compared with parent strain E. coli FB-04(pta1). Thus, mutation of the genomic of ptsH gene provided an alternative method to weaken the PTS and improve the efficiency of carbon source utilization.     

Rational design and analysis of an <Emphasis Type="Italic">Escherichia coli</Emphasis> strain for high-efficiency tryptophan production

l-tryptophan (l-trp) is a precursor of various bioactive components and has great pharmaceutical interest. However, due to the requirement of several precursors and complex regulation of the pathways involved, the development of an efficient l-trp production strain is challenging. In this study, Escherichia coli (E. coli) strain KW001 was designed to overexpress the l-trp operator sequences (trpEDCBA) and 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (aroG ^fbr ). To further improve the production of l-trp, pyruvate kinase (pykF) and the phosphotransferase system HPr (ptsH) were deleted after inactivation of repression (trpR) and attenuation (attenuator) to produce strain KW006. To overcome the relatively slow growth and to increase the transport rate of glucose, strain KW018 was generated by combinatorial regulation of glucokinase (galP) and galactose permease (glk) expression. To reduce the production of acetic acid, strain KW023 was created by repressive regulation of phosphate acetyltransferase (pta) expression. In conclusion, strain KW023 efficiently produced 39.7 g/L of l-trp with a conversion rate of 16.7% and a productivity of 1.6 g/L/h in a 5 L fed-batch fermentation system.     

Studies in the taxonomy of the Polypores II

The following genera are redefined:Albatrellus S. F. Gray,Heterobasidion Bref.,Haploporus Bond. et Sing. ex Sing.,Fomitopsis P. Karst. andRigidoporus Murrill two new subgenera are described:Polyporus subgen.Dendropolyporus Pouz. (type:Polyporus umbellatus) andRigidoporus subgen.Neooxyporus Pouz. (type:Polyporus latemarginatus); the genusOxyporus (Bourd. etGalz.)Donk is classified as a subgenus of the genusRigidoporus,Murrill and the generaBjerkandera P. Karst. andLeptoporus quél. are classified as subgenera of the genusTyromyces P. Karst. The new subfamilyAlbatrelloideae Pouz. (genera:Albatrellus andGrifola) is described and 14 new specific combinations are made. The new genusIrpicodon Pouz. (type:Irpex pendulus) is proposed.     

Effect of Active Site Pocket Structure Modification of <Emphasis Type="SmallCaps">d</Emphasis>-Stereospecific Amidohydrolase on the Recognition of Stereospecific and Hydrophobic Substrates

d-Stereospecific amidohydrolase (DAH) from Streptomyces sp. 82F2 has potential utility for the synthesis of d/l configuration dipeptides by an aminolysis reaction. Structural comparison of DAH with substrate-bound d-amino acid amidase revealed that three residues located in the active site pocket of DAH (Thr145, Ala267, and Gly271) might be involved in interactions with d-phenylalanine substrate. We substituted Ala267 and Gly271, which are located at the bottom of the hydrophobic pocket of DAH, with Phe and observed changes in the stereoselectivity and specific activity toward the free and acetylated forms of d/l-Phe-methyl esters. In contrast, the mutation of Thr145, which likely supplies negative charge for recognition of the amino group of the substrate, hardly affected the stereoselectivity of the enzyme. A similar effect was observed in an investigation of hydrolysis and aminolysis reactions using the acetylated forms of d/l-Phe-methyl esters and 1,8-diaminooctane as an acyl-donor and acyl-acceptor, respectively. Substrate binding by DAH was disrupted by the mutation of Ala267 to Val or Trp and kinetic analysis showed that the hydrophobicity of the bottom of the active site pocket (Ala267 and Gly271) is important for both stereoselectivity and recognition of hydrophobic substrates.     

Engineering rTCA pathway and C4-dicarboxylate transporter for <Emphasis Type="SmallCaps">l</Emphasis>-malic acid production

l-Malic acid is an important component of a vast array of food additives, antioxidants, disincrustants, pharmaceuticals, and cosmetics. Here, we presented a pathway optimization strategy and a transporter modification approach to reconstruct the l-malic acid biosynthesis pathway and transport system, respectively. First, pyruvate carboxylase (pyc) and malate dehydrogenase (mdh) from Aspergillus flavus and Rhizopus oryzae were combinatorially overexpressed to construct the reductive tricarboxylic acid (rTCA) pathway for l-malic acid biosynthesis. Second, the l-malic acid transporter (Spmae) from Schizosaccharomyces pombe was engineered by removing the ubiquitination motification to enhance the l-malic acid efflux system. Finally, the l-malic acid pathway was optimized by controlling gene expression levels, and the final l-malic acid concentration, yield, and productivity were up to 30.25 g L^?1, 0.30 g g^?1, and 0.32 g L^?1 h^?1 in the resulting strain W4209 with CaCO₃ as a neutralizing agent, respectively. In addition, these corresponding parameters of pyruvic acid remained at 30.75 g L^?1, 0.31 g g^?1, and 0.32 g L^?1 h^?1, respectively. The metabolic engineering strategy used here will be useful for efficient production of l-malic acid and other chemicals.     

Differentiation of species in Gonioloboceras

The type specimen ofGonioloboceras goniolobum (Meek), rediscovered by Spath in the British Museum, is the foundation for a more accurate comparative study of this and other species ofGonioloboceras.Gonioloboceras described asG. goniolobum byElias in 1938 is differentiated asGonioloboceras schmidti, new species. Suture sets (new term) for several growth stages inG. goniolobum (Meek),G. welleriSmith,G. schmidtiElias, G.eliasiMiller &Owen, andG. asiaticumLibrovitch are assembled and used for differentiation of the species.The Kazakhstan goniatite faunule containingG. asiaticum is considered of very late Pennsylvanian age.     

Flechtensystematische Studien IV. Die GattungGyalidea Lett

Im Artikel wird die ursprüngliche Gattungsdiagnose derGyalidea Lett. um die Merkmale der Pilzkomponente ergänzt. Die angenommenen verwandtschaftlichen Beziehungen derGyalidea zu der FamilieAsterothyriaceae werden diskutiert. Alle bisher festgestellten Arten mit Übersichten der revidierten Proben werden kurz beschrieben. Neue Taxa und Kombinationen:Gyalidea dodgei spec. n.,G. epiphylla spec. n.,G. mayaguezensis spec. n.,G. portoricensis spec. n.,G. fritzei (Stein) comb. n.,G. fritzei var.rivularis (Eitn.) comb. n.,G. hyalinescens (Nyl.) comb. n.,G. lecideopsis var.convarians (Nyl.) comb. n.,G. lecideopsis var.stigmatoides (Nyl.) comb. n.,G. mexicana (B. de Lesd.) comb. n. undG. subscutellaris (Vězda) comb. n.     

Stress-inducible overexpression of <Emphasis Type="Italic">glyoxalase I</Emphasis> is preferable to its constitutive overexpression for abiotic stress tolerance in transgenic <Emphasis Type="Italic">Brassica juncea</Emphasis>

The glyoxalase system catalyzes the conversion of cytotoxic methylglyoxal to d-lactate via the intermediate S-d-lactoylglutathione. It comprises two enzymes, Glyoxalase I (Gly I) and Glyoxalase II (Gly II), and reduced glutathione which acts as a cofactor by anchoring the substrates in the active sites of the two enzymes. The overexpression of both Gly I and Gly II, either alone or in combination, has earlier been reported to confer tolerance to multiple abiotic stresses. In the present study, we sought to evaluate the consequences of constitutive and stress-induced overexpression of Gly I on the performance and productivity of plants. Towards this end, several Gly I transgenic Brassica juncea lines (designated as R and S lines) were generated in which the glyoxalase I (gly I) gene was expressed under the control of either a stress-inducible rd29A promoter or a constitutive CaMV 35S promoter. Both the R and S lines showed enhanced tolerance to salinity, heavy metal, and drought stress when compared to untransformed control plants. However, the S lines showed yield penalty under non-stress conditions while no such negative effect was observed in the R lines. Our results indicate that the overexpression of the gly I gene under the control of stress-inducible rd29A promoter is a better option for improving salt, drought and heavy metal stress tolerance in transgenic plants.     

Characterization of a novel asparaginase from soil metagenomic libraries generated from forest soil

Objectives

To screen soil metagenomic libraries for novel enzymes with enhanced activities.

Results

To screen soil metagenomic libraries for novel enzymes with enhanced activities. A novel l-asparaginase was identified from forest soil metagenome and its characteristics were studied. The purified protein had a specific activity of 696 IU mg^?1 and optimum activity at pH 7 and 35 °C. Enhanced enzyme activities were observed in the presence of Mg²⁺, Ca²⁺ and K⁺. The K_m value, 2 mM, and enzyme specificity constant 7.7 mM^?1s^?1 indicated that the recombinant enzyme has good substrate affinity to l-asparagine compared with commercially-available Escherichia coli asparaginase. The IC₅₀ value of 0.78 µg ml^?1 (0.47 IU ml^?1) was observed with HL60 cell line and 0.39 µg ml^?1(0.23 IU ml^?1) with MOLT-3 and MOLT-4 cell lines, which is better than that of commercially-available drugs.

Conclusion

The soil metagenome derived l-asparaginase with enhanced activities could be a potential candidate to develop as a drug in Acute Lymphoblastic Leukemia (ALL) therapy.

     

Beiträge zur Sedimentation und fossilführung des hunsrückschiefers 14.Viriatellina fuchsi (Kutscher, 1931) im Hunsrückschiefer und im Tentaculitenknollenkalk thüringens

The new genusViriatellina Bou?ek, 1964 unites the two older species namedNovakia gemündina Runzheimer, 1932 andTentaculites fuchsi Kutscher, 1931. ThusViriatellina gemuendina (Runzheimer) is cancelled, on the other handViriatellina fuchsi (Kutscher) remains valid. RecentlyViriatellina fuchsi (Kutscher) has been found in the tentaculitiferous limestone of Thuringia.     

Microbial production of astilbin,a bioactive rhamnosylated flavanonol,from taxifolin

Flavonoids are plant-based polyphenolic biomolecules with a wide range of biological activities. Glycosylated flavonoids have drawn special attention in the industries as it improves solubility, stability, and bioactivity. Herein, we report the production of astilbin (ATN) from taxifolin (TFN) in genetically-engineered Escherichia coli BL21(DE3). The exogenously supplied TFN was converted to ATN by 3-O-rhamnosylation utilizing the endogeneous TDP-l-rhamnose in presence of UDP-glycosyltransferase (ArGT3, Gene Bank accession number: At1g30530) from Arabidopsis thaliana. Upon improving the intracellular TDP-l-rhamnose pool by knocking out the chromosomal glucose phosphate isomerase (pgi) and d-glucose-6-phosphate dehydrogenase (zwf) deletion along with the overexpression of rhamnose biosynthetic pathway increases the biotransformation product, ATN with total conversion of ~49.5?±?1.67% from 100 µM of taxifolin. In addition, the cytotoxic effect of taxifolin-3-O-rhamnoside on PANC-1 and A-549 cancer cell lines was assessed for establishing ATN as potent antitumor compound.     

The variability of critical species ofLotus L. in Iran and in the neighbouring countries I. the circle ofL. corniculatus L. andL. gebelia Vent.

The region of Iran, Iraq, Afghanistan and the neighbouring countries is important for some groups of the speciesLotus L., especially those of the circle ofL. corniculatus L. andL. gebelia Vent. The first group is represented by the speciesL. corniculatus L. with 4 subspecies (3 of which are important for this region), andL. tenuis Waldst. etKit. which here attains the eastern boundary of the continuous area of distribution, and by the eastern speciesL. krylovii Schischk. etSerg. andL. rechingeri Chrtková-?ertová. The second group is represented by the speciesL. gebelia Vent.,L. michauxianus Ser. in DC. andL. libanoticus Boiss. their areas of distribution covering mostly those regions. Most of the species show considerable variability within the species.     

<Emphasis Type="Italic">C7</Emphasis>-prenylation of tryptophanyl and <Emphasis Type="Italic">O</Emphasis>-prenylation of tyrosyl residues in dipeptides by an <Emphasis Type="Italic">Aspergillus terreus</Emphasis> prenyltransferase

During our search for novel prenyltransferases, a putative gene ATEG_04218 from Aspergillus terreus raised our attention and was therefore amplified from strain DSM 1958 and expressed in Escherichia coli. Biochemical investigations with the purified recombinant protein and different aromatic substrates in the presence of dimethylallyl diphosphate revealed the acceptance of all the tested tryptophan-containing cyclic dipeptides. Structure elucidation of the main enzyme products by NMR and MS analyses confirmed the attachment of the prenyl moiety to C-7 of the indole ring, proving the identification of a cyclic dipeptide C7-prenyltransferase (CdpC7PT). For some substrates, reversely C3- or N1-prenylated derivatives were identified as minor products. In comparison to the known tryptophan-containing cyclic dipeptide C7-prenyltransferase CTrpPT from Aspergillus oryzae, CdpC7PT showed a much higher substrate flexibility. It also accepted cyclo-l-Tyr-l-Tyr as substrate and catalyzed an O-prenylation at the tyrosyl residue, providing the first example from the dimethylallyltryptophan synthase (DMATS) superfamily with an O-prenyltransferase activity towards dipeptides. Furthermore, products with both C7-prenyl at tryptophanyl and O-prenyl at tyrosyl residue were detected in the reaction mixture of cyclo-l-Trp-l-Tyr. Determination of the kinetic parameters proved that (S)-benzodiazepinedione consisting of a tryptophanyl and an anthranilyl moiety was accepted as the best substrate with a K _M value of 204.1 μM and a turnover number of 0.125 s^?1. Cyclo-l-Tyr-l-Tyr was accepted with a K _M value of 1,411.3 μM and a turnover number of 0.012 s^?1.     

Broad-specificity amino acid racemase,a novel non-antibiotic selectable marker for transgenic plants

The broad-specificity amino acid racemase (Bsar) from Pseudomonas putida catalyzes the racemization of various amino acids, offering a flexible and feasible platform to develop a new non-antibiotic selectable marker system for plant transformation. In the present study, we demonstrated that a Bsar variant, Bsar-R174K, that is useful as a selectable marker gene in Arabidopsis and rice that were susceptible to l-lysine and D-alanine. The introduction of wild-type Bsar, Bsar-R174K or Bsar-R174A into E. coli lysine or asparagine auxotrophs was able to rescue the growth of these microorganisms in minimal media supplemented with selectable amino acid enantiomers. The transformation of Arabidopsis with Bsar or Bsar variants based on d-alanine selection revealed that Bsar-R174K had the greatest efficiency (2.40%), superior to kanamycin selection-based transformation (1.10%). Whereas, l-lysine-based selection exhibited lower efficiency for Bsar-R174K (0.17%). The progenies of selected Bsar-R174K transgenic Arabidopsis revealed normal growth properties. In addition, Bsar-R174K transgenic rice was obtained on l-lysine medium with an efficiency of 0.9%, and the progenies of the transgenic rice revealed morphologically normal phenotypes comparable with their wild-type counterparts. This study presents the first report of broad range amino acid racemase Bsar-R174K as a non-antibiotic selectable marker system applied in transgenic plants.     

Efficient production of indigoidine in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Indigoidine is a bacterial natural product with antioxidant and antimicrobial activities. Its bright blue color resembles the industrial dye indigo, thus representing a new natural blue dye that may find uses in industry. In our previous study, an indigoidine synthetase Sc-IndC and an associated helper protein Sc-IndB were identified from Streptomyces chromofuscus ATCC 49982 and successfully expressed in Escherichia coli BAP1 to produce the blue pigment at 3.93 g/l. To further improve the production of indigoidine, in this work, the direct biosynthetic precursor l-glutamine was fed into the fermentation broth of the engineered E. coli strain harboring Sc-IndC and Sc-IndB. The highest titer of indigoidine reached 8.81 ± 0.21 g/l at 1.46 g/l l-glutamine. Given the relatively high price of l-glutamine, a metabolic engineering technique was used to directly enhance the in situ supply of this precursor. A glutamine synthetase gene (glnA) was amplified from E. coli and co-expressed with Sc-indC and Sc-indB in E. coli BAP1, leading to the production of indigoidine at 5.75 ± 0.09 g/l. Because a nitrogen source is required for amino acid biosynthesis, we then tested the effect of different nitrogen-containing salts on the supply of l-glutamine and subsequent indigoidine production. Among the four tested salts including (NH₄)₂SO₄, NH₄Cl, (NH₄)₂HPO₄ and KNO₃, (NH₄)₂HPO₄ showed the best effect on improving the titer of indigoidine. Different concentrations of (NH₄)₂HPO₄ were added to the fermentation broths of E. coli BAP1/Sc-IndC+Sc-IndB+GlnA, and the titer reached the highest (7.08 ± 0.11 g/l) at 2.5 mM (NH₄)₂HPO₄. This work provides two efficient methods for the production of this promising blue pigment in E. coli.