Characterization of<Emphasis Type="SmallCaps"> D</Emphasis>-lactic acid,spore-forming bacteria and <Emphasis Type="Italic">Terrilactibacillus laevilacticus</Emphasis> SK5-6 as potential industrial strains

In this study, we screened and isolated D-lactic acid-producing bacteria from soil and tree barks collected in Thailand. Among the isolates obtained, Terrilactibacillus laevilacticus SK5-6 exhibited good D-lactate production in the primary screening fermentation (99.27 g/L final lactate titer with 0.90 g/g yield, 1.38 g/L?h, and 99.00% D-enantiomer equivalent). Terrilactibacillus laevilacticus SK5-6 is a Gram-positive, endospore-forming, homofermentative D-lactate producer that can ferment a wide range of sugars to produce D-lactate. Unlike the typical D-lactate producers, such as catalase-negative Sporolactobacillus sp., T. laevilacticus SK5-6 possesses catalase activity; therefore, a two-phase fermentation was employed for D-lactate production. During an aerobic preculture stage, a high-density cell mass was rapidly obtained due to aerobic respiration. When transferred to the fermentation stage at the correct physiological stage (inoculum age) and proper concentration of cell mass (inoculum size), T. laevilacticus rapidly converted glucose into D-lactate under anaerobic conditions, resulting in a high final lactate titer (102.22 g/L), high yield (0.84 g/g), and high productivity (2.13 g/L?h). When the process conditions were shifted from an aerobic to an anaerobic environment, unlike other lactate-producing bacteria, the mixed acid fermentation route was not activated in the culture of T. laevilacticus SK5-6 during the fermentation stage when some trace oxygen still remained. Our study demonstrates the excellent characteristics of this isolate for D-lactate production; in particular, a high product yield was obtained without byproduct formation. Based on these key characteristics of T. laevilacticus SK5-6, we suggest that this isolate is a novel D-lactate producer for use in industrial fermentation.     

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.     

Characterization of aspartate kinase and homoserine dehydrogenase from <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> IWJ001 and systematic investigation of <Emphasis Type="SmallCaps">l</Emphasis>-isoleucine biosynthesis

Previously we have characterized a threonine dehydratase mutant TD^F383V (encoded by ilvA1) and an acetohydroxy acid synthase mutant AHAS^{P176S, D426E, L575W} (encoded by ilvBN1) in Corynebacterium glutamicum IWJ001, one of the best l-isoleucine producing strains. Here, we further characterized an aspartate kinase mutant AK^A279T (encoded by lysC1) and a homoserine dehydrogenase mutant HD^G378S (encoded by hom1) in IWJ001, and analyzed the consequences of all these mutant enzymes on amino acids production in the wild type background. In vitro enzyme tests confirmed that AK^A279T is completely resistant to feed-back inhibition by l-threonine and l-lysine, and that HD^G378S is partially resistant to l-threonine with the half maximal inhibitory concentration between 12 and 14 mM. In C. glutamicum ATCC13869, expressing lysC1 alone led to exclusive l-lysine accumulation, co-expressing hom1 and thrB1 with lysC1 shifted partial carbon flux from l-lysine (decreased by 50.1 %) to l-threonine (4.85 g/L) with minor l-isoleucine and no l-homoserine accumulation, further co-expressing ilvA1 completely depleted l-threonine and strongly shifted carbon flux from l-lysine (decreased by 83.0 %) to l-isoleucine (3.53 g/L). The results demonstrated the strongly feed-back resistant TD^F383V might be the main driving force for l-isoleucine over-synthesis in this case, and the partially feed-back resistant HD^G378S might prevent the accumulation of toxic intermediates. Information exploited from such mutation-bred production strain would be useful for metabolic engineering.     

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.     

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.     

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.     

Some chromosome numbers of the CzechoslovakLeguminosae

Chromosome numbers of the Czechoslovak species of the genusLotus, from various localities have been determined. The paper includes the speciesLotus uliginosus Schkuhr,L.tenuis Waldst. etKit. andL. borbásii Ujhelyi.     

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.     

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.     

Glacial relics in the bryoflora of the highlands Českomoravská vrchovina (Bohemian-Moravian Highlands); Their habitat and cenotaxonomic value

The paper sums up the information which the author has so far collected about the habitats and phytocenotic conditions of the glacial relics ofScorpidium scorpioides (Hedw.) Limpr.,Calliergon trifarium (Web. etMohr.)Kindb.,Paludella squarrosa (Hedw.) Brid., andMeesia triquetra (Hook. etTayl.)Aongstr. in the Highlands ?eskomoravská vrchovina (Bohemian-Moravian Highlands) in Czechoslovakia. Simultaneously, it presents a survey of the localities in which the occurrence of these species has been ascertained up to now.     

Über böhmische und türkische Vertreter vonAnetoceras (Ammon., Unterdevon)

The Bohemian speciesAnetoceras solitarius (Barrande 1865) is re-described, including the TurkishA. barrandei (De Verneuil 1866). The other Bohemian species areA. aff.advolvens Erben 1960 and?A. fritschi (Barrande 1877). A new speciesA. recticostatum n. sp. is described from the German Lower Devonian, and several still problematical species of the genus are discussed. Finally, the supposed phylogenetic position and the relations ofAnetoceras are pointed out. The Upper SiegenianA. hunsrueckianum Erben 1960 is regarded as most primitive coiled ammonoid and as connecting link between the bactritids, and early coiled ammonoids.Mimosphinctes Eichenberg is considered to be a descendant fromAnetoceras.     

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.     

Karyo-taxonomic study of the genus<Emphasis Type="Italic">Pseudolysimachion</Emphasis> (<Emphasis Type="Italic">Scrophulariaceae</Emphasis>) in the Czech Republic and Slovakia

Flow cytometry was used to determine ploidy levels in the Czech and Slovak taxa of the genusPseudolysimachion (W.D.J. Koch)Opiz (=Veronica auct. p.p.,Scrophulariaceae). In total, 123 populations from the Czech Republic, Slovakia, Ukraine (one locality), Austria (one locality) and Hungary (one locality) were analyzed. InP. maritimum (L.)Á. Löve etD. Löve andP. spicatum (L.)Opiz, two cytotypes were found: diploid (2n=2x=34) and tetraploid (2n=4x=68). In both species the tetraploid cytotype predominated (P. maritimum: 41 tetraploid populations out of 45;P. spicatum: 57 tetraploid populations out of 58). The two cytotypes ofP. maritimum have no taxonomic significance because ploidy level is not obviously correlated with morphology, distribution pattern or ecology. Tetraploid populations ofP. spicatum belong to two morphologically different subspecies, subsp.spicatum and subsp.fischeri Trávní?ek. The diploid cytotype (one population only) should be provisionally classified as a third subspecies ofP. spicatum, which is morphologically similar to the Asian subsp.porphyrianum (Pavlov)Trávní?ek. Only diploid plants (2n=2x=34) ofP. orchideum (Crantz)Wraber were found; all 13 populations that were analyzed belong toP. orchideum s.str. One diploid population sample ofP. spurium subsp.foliosum (Waldst. etKit.)Holub (2n=2x=34) and one tetraploid sample ofP. incanum subsp.pallens (Host)Trávní?ek (2n=4x=68) were also analyzed. In addition, three tetraploid populations of hybrid origin were investigated:P. maritimum ×P. spicatum subsp.spicatum (one population) andP. maritimum ×P. spurium subsp.foliosum (two populations). While hybrid plants ofP. maritimum ×P. spicatum arose from tetraploid parental species, plants ofP. maritimum ×P. spurium probably resulted from a cross between tetraploidP. maritimum and diploidP. spurium. The putative origin and evolutionary importance of polyploids in thePseudolysimachion are discussed.     

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.     

Glacial relics in the bryoflora of the highlands Českomoravská vrchovina (Bohemian-Moravian Highlands); Their habitat and cenotaxonomic value

The paper sums up the information which the author has so far collected about the habitats and phytocenotic conditions of the glacial relics ofScorpidium scorpioides (Hedw.) Limpr.,Calliergon trifarium (Web. etMohr.) Kindb.,Paludella squarrosa (Hedw.) Bred., andMeesia triquetra (Hook. etTayl.) Aongstr. in the Highlands ?eskomoravská vrchovina (Bohemian-Moravian Highlands) in Czechoslovakia. Simultaneously, it presents a survey of the localities in which the occurrence of these species has been ascertained up to now.     

<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.     

Characterization of an α-amino-ɛ-caprolactam racemase with broad substrate specificity from <Emphasis Type="Italic">Citreicella</Emphasis> sp. SE45

α-Amino-ε-caprolactam (ACL) racemizing activity was detected in a putative dialkylglycine decarboxylase (EC 4.1.1.64) from Citreicella sp. SE45. The encoding gene of the enzyme was cloned and transformed in Escherichia coli BL21 (DE3). The molecular mass of the enzyme was shown to be 47.4 kDa on SDS–polyacrylamide gel electrophoresis. The enzymatic properties including pH and thermal optimum and stabilities were determined. This enzyme acted on a broad range of amino acid amides, particularly unbranched amino acid amides including l-alanine amide and l-serine amide with a specific activity of 17.5 and 21.6 U/mg, respectively. The K _m and V _max values for d- and l-ACL were 5.3 and 2.17 mM, and 769 and 558 μmol/min.mg protein, respectively. Moreover, the turn over number (K _cat) and catalytic efficiency (K _cat/K _m ) of purified ACL racemase from Citreicella sp. SE45 using l-ACL as a substrate were 465 S^?1 and 214 S^?1mM^?1, respectively. The new ACL racemase from Citreicella sp. SE45 has a potential to be used as the biocatalytic application.     

Enantiomer-Selective Photo-Induced Reaction of Protonated Tryptophan with Disaccharides in the Gas Phase

In order to investigate chemical evolution in interstellar molecular clouds, enantiomer-selective photo-induced chemical reactions between an amino acid and disaccharides in the gas phase were examined using a tandem mass spectrometer containing an electrospray ionization source and a cold ion trap. Ultraviolet photodissociation mass spectra of cold gas-phase noncovalent complexes of protonated tryptophan (Trp) enantiomers with disaccharides consisting of two d-glucose units, such as d-maltose or d-cellobiose, were obtained by photoexcitation of the indole ring of Trp. NH₂CHCOOH loss via cleavage of the C_α–C_β bond in Trp induced by hydrogen atom transfer from the NH₃ ⁺ group of a protonated Trp was observed in a noncovalent heterochiral H⁺(l-Trp)(d-maltose) complex. In contrast, a photo-induced chemical reaction forming the product ion with m/z 282 occurs in homochiral H⁺(d-Trp)(d-maltose). For d-cellobiose, both NH₂CHCOOH elimination and the m/z 282 product ion were observed, and no enantiomer-selective phenomena occurred. The m/z 282 product ion indicates that the photo-induced C-glycosylation, which links d-glucose residues to the indole moiety of Trp via a C–C bond, can occur in cold gas-phase noncovalent complexes, and its enantiomer-selectivity depends on the structure of the disaccharide.     

New subfamily Proschizophoriinae of dalmanellid Brachiopds

Proschizophoria Maillieux is restudied and found to be related to the Silurian genusIdiorthis McLearn and a new genusCordatomyonia, type speciesCordatomyonia shupei, n. sp.Proschizophoria, Idiorthis, andCordatomyonia are united in the new subfamily Proschizophoriinae which is assigned to the dalmanellid family Rhipidomellidae. Rhipidomellidae and its included subfamilies Heterorthinae, Rhipidomellinae, and Proschizophoriinae are emended and newly diagnosed. A new dalmanellid genus,Megasalopina, is proposed for the Lower Devonian species“Dalmanella” elevata Williams & Breger and its allies. It is thought to have been derived from the draboviinid genusSalopina Boucot and therefore belongs to the Schizophoriidae.Dalmanellopsis Khalfin is removed from the synonymy ofLevenea and is shown to be related toSalopina.