Enantioselective hydrolysis of racemic styrene oxide by epoxide hydrolase of<Emphasis Type="Italic">Rhodosporidium kratochvilovae</Emphasis> SYU-08

Enantioselective hydrolysis for the production of chiral styrene oxide was investigated using the epoxide hydrolase activity of a newly isolatedRhodosporidium kratochvilovae SYU-08. The effects of reaction prameters—buffer type, pH, temperature, initial substrate concentrations, phenyl-1,2-ethanediol concentrations on hydrolysis rate, and enantioselectivity—were analyzed. Optically active (S)-styrene oxide with an enantiomeric excess higher than 99 % was obtained from its racemate with a yield of 38 % (theoretically 50% maximum yield) from an initial concentration of 80 mM.     

Biosynthesis of (R)-phenyl-1,2-ethanediol from racemic styrene oxide by using bacterial and marine fish epoxide hydrolases

Enantio-convergent hydrolysis of racemic styrene oxides was achieved to prepare enantiopure (R)-phenyl-1,2-ethanediol by using two recombinant epoxide hydrolases (EHs) of a bacterium, Caulobacter crescentus, and a marine fish, Mugil cephalus. The recombinant C. crescentus EH primarily attacked the benzylic carbon of (S)-styrene oxide, while the M. cephalus EH preferentially attacked the terminal carbon of (R)-styrene oxide, thus leading to the formation of (R)-phenyl-1,2-ethanediol as the main product. (R)-Phenyl-1,2-ethanediol was obtained with 90% enantiomeric excess and yield as high as 94% from 50 mM racemic styrene oxides in a one-pot process.     

Diastereoselective synthesis of optically active (2<Emphasis Type="Italic">R</Emphasis>,5<Emphasis Type="Italic">R</Emphasis>)-hexanediol

Diastereoselective reduction of diketones with Lactobacillus kefir DSM 20587 was examined. The reduction of both oxo-functions proceeded highly diastereoselectively. (2 R,5 R)-Hexanediol 3 was produced starting from (2,5)-hexanedione 1 in quantitative yields with enantiomeric excess >99% and diastereomeric excess >99%. The reaction conditions were optimized: maximum yield of (2 R,5 R)-hexanediol was reached at pH 6, 30 degrees C and with equal amounts of substrate and cosubstrate. The applicability of the system in fed-batch experiments was demonstrated. The feed specific biomass concentration required to reach maximal yield and selectivity in fed-batch mode was determined.     

<Emphasis Type="Italic">R</Emphasis>-acetoin accumulation and dissimilation in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

Klebsiella pneumoniae is a 2,3-butanediol producer, and R-acetoin is an intermediate of 2,3-butanediol production. R-acetoin accumulation and dissimilation in K. pneumoniae was studied here. A budC mutant, which has lost 2,3-butanediol dehydrogenase activity, accumulated high levels of R-acetoin in culture broth. However, after glucose was exhausted, the accumulated R-acetoin could be reused by the cells as a carbon source. Acetoin dehydrogenase enzyme system, encoded by acoABCD, was responsible for R-acetoin dissimilation. acoABCD mutants lost the ability to grow on acetoin as the sole carbon source, and the acetoin accumulated could not be dissimilated. However, in the presence of another carbon source, the acetoin accumulated in broth of acoABCD mutants was converted to 2,3-butanediol. Parameters of R-acetoin production by budC mutants were optimized in batch culture. Aerobic culture and mildly acidic conditions (pH 6–6.5) favored R-acetoin accumulation. At the optimized conditions, in fed-batch fermentation, 62.3 g/L R-acetoin was produced by budC and acoABCD double mutant in 57 h culture, with an optical purity of 98.0 %, and a substrate conversion ratio of 28.7 %.     

Production of (<Emphasis Type="Italic">R</Emphasis>)-ethyl-3,4-epoxybutyrate by newly isolated <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> containing epoxide hydrolase

Several new microorganisms have been isolated from soil samples with high epoxide hydrolase activity toward ethyl 3,4-epoxybutyrate. Screening was performed by enrichment culture on alkenes as sole carbon source, followed by chiral gas chromatography. Eight strains were discovered with enantioselectivity from moderate to high level and identified as bacterial and yeast species. Cells were cultivated under aerobic condition at 30°C using glucose as carbon source and resting cells were used as biocatalysts for kinetic resolution of ethyl 3,4-epoxybutyrate. Among isolated microorganisms, Acinetobacter baumannii showed highest enantioselectivity for (S)-enantiomer, resulting in (R)-ethyl-3,4-epoxybutyrates (>99%ee, 46% yield). It is the first report on the fact that epoxide hydrolases originating from bacterial species of A. baumannii was applied to kinetic resolution of ethyl 3,4-epoxybutyrate in order to obtain enantiopure high-value-added (R)-ethyl-3,4-epoxybutyrate.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Production of epoxide hydrolases in batch fermentations of <Emphasis Type="Italic">Botryosphaeria rhodina</Emphasis>

The filamentous fungus Botryosphaeria rhodina (ATCC 9055) was investigated related to its ability for epoxide hydrolase (EH) production. Epoxide hydrolase activity is located at two different sites of the cells. The larger part is present in the cytosol (70%), while the smaller part is associated to membranes (30%). In media optimization experiments, an activity of 3.5 U/gDW for aromatic epoxide hydrolysis of para-nitro-styrene oxide (pNSO) could be obtained. Activity increased by 30% when pNSO was added to the culture during exponential growth. An increase of enzyme activity up to 6 U/gDW was achieved during batch-fermentations in a bioreactor with 2.7 l working volume. Evaluation of fermentations with 30 l working volume revealed a relation of oxygen uptake rate to EH expression. Oxygen limitation resulted in a decreased EH activity. Parameter estimation by the linearization method of Hanes yielded Km values of 2.54 and 1.00 mM for the substrates S-pNSO and R-pNSO, respectively. vmax was 3.4 times higher when using R-pNSO. A protein purification strategy leading to a 47-fold increase in specific activity (940 U/mgProtein) was developed as a first step to investigate molecular and structural characteristics of the EH.     

Functional expression and magnetic nanoparticle-based Immobilization of a protein-engineered marine fish epoxide hydrolase of <Emphasis Type="Italic">Mugil cephalus</Emphasis> for enantioselective hydrolysis of racemic styrene oxide

A triple-point mutated fish microsomal epoxide hydrolase (mEH) gene from Mugil cephalus was expressed in Escherichia coli in the presence of various chaperones to prevent protein aggregations. The enantioselective hydrolytic activity was more than doubled by co-expressing the EH mutant gene with pGro7 plasmid. The highly active EH mutant with a his-tag was immobilized onto magnetic silica assembled with NiO nanoparticles. The immobilized mEH mutant was re-used more than 10 times with less than 10% activity loss. (S)-Styrene oxide with 98% enantiopurity was repeatedly obtained with over 50% of the theoretical yield by the magnetically separable high-performance mEH mutant.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Fine genetic mapping localizes cucumber scab resistance gene <Emphasis Type="Italic">Ccu</Emphasis> into an <Emphasis Type="Italic">R</Emphasis> gene cluster

Scab, caused by Cladosporium cucumerinum, is an important disease of cucumber, Cucumis sativus. In this study, we conducted fine genetic mapping of the single dominant scab resistance gene, Ccu, with 148 F₉ recombinant inbred lines (RILs) and 1,944 F₂ plants derived from the resistant cucumber inbred line 9110Gt and the susceptible line 9930, whose draft genome sequence is now available. A framework linkage map was first constructed with simple sequence repeat markers placing Ccu into the terminal 670 kb region of cucumber Chromosome 2. The 9110Gt genome was sequenced at 5× genome coverage with the Solexa next-generation sequencing technology. Sequence analysis of the assembled 9110Gt contigs and the Ccu region of the 9930 genome identified three insertion/deletion (Indel) markers, Indel01, Indel02, and Indel03 that were closely linked with the Ccu locus. On the high-resolution map developed with the F₂ population, the two closest flanking markers, Indel01 and Indel02, were 0.14 and 0.15 cM away from the target gene Ccu, respectively, and the physical distance between the two markers was approximately 140 kb. Detailed annotation of the 180 kb region harboring the Ccu locus identified a cluster of six resistance gene analogs (RGAs) that belong to the nucleotide binding site (NBS) type R genes. Four RGAs were in the region delimited by markers Indel01 and Indel02, and thus were possible candidates of Ccu. Comparative DNA analysis of this cucumber Ccu gene region with a melon (C. melo) bacterial artificial chromosome (BAC) clone revealed a high degree of micro-synteny and conservation of the RGA tandem repeats in this region.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Biotransformation of racemic diisophorone by <Emphasis Type="Italic">Cephalosporium aphidicola</Emphasis> and <Emphasis Type="Italic">Neurospora crassa</Emphasis>

Racemic diisophorone (500 mg) was converted by Cephalosporium aphidicola and Neurospora crassa over 10 days at 25 °C to 8β-hydroxydiisophorone in yields of 10% (52 mg) and 20% (103 mg), respectively. The structure was established by IR, specific rotation, mass spectral, 1D and 2D-NMR studies.Revisions requested 2 March 2005 and 21 April 2005; Revisions received 8 April 2005 and 10 May 2005     

Stereoselective oxidation of racemic 1-arylethanols by basil cultured cells of <Emphasis Type="Italic">Ocimum </Emphasis><Emphasis Type="Italic">basilicum</Emphasis> cv. <Emphasis Type="Italic">Purpurascens</Emphasis>

The biotransformation of racemic 1-phenylethanol (30 mg) with plant cultured cells of basil (Ocimum basilicum cv. Purpurascens, 5 g wet wt) by shaking 120 rpm at 25°C for 7 days in the dark gave (R)-(+)-1-phenylethanol and acetophenone in 34 and 24% yields, respectively. The biotransformation can be applied to other 1-arylethanols and basil cells oxidized the (S)-alcohols to the corresponding ketones remaining the (R)-alcohols in excellent ee.     

Molecular analysis shows differential expression of <Emphasis Type="Italic">R</Emphasis>-<Emphasis Type="Italic">spondin1</Emphasis> in zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) gonads

R-spondin1 (RSPO1) is a potential female-determining gene in human (Homo sapiens) and mouse (Mus musculus). Its differential expression in these mammals is correlated with signaling for sex determination. As a way of studying sex determination in fish we cloned and analyzed a RSPO1 gene in zebrafish (Danio rerio). Using real-time PCR, we observed that RSPO1 is expressed more strongly in ovaries than in testes, suggesting that RSPO1 may have a role in gonad differentiation. High RSPO1 expression was detected in some non-gonadal organs like muscle and kidneys. In situ hybridization results demonstrate that RSPO1 is expressed in premature germ cells, in oogonia and primary oocytes in ovaries and in spermatogonia and spermatocytes in testes. It is also expressed in gonad somatic cells during gonadal development: in granulosa cells and theca cells of early and late cortical-alveolar stage follicles in ovaries, and in Leydig cells in testes. This differential expression may indicate that RSPO1 has a role(s) in zebrafish gonad development and differentiation. By fusing zebrafish RSPO1 with a green fluorescent protein gene, we found that RSPO1 is located in the cytosol and Golgi apparatus but not the nucleus of fish epithelioma papulosum cyprinid (EPC) cells. These preliminary findings suggest some aspects of RSPO1 like differential expression linked to sex determination may be conserved in fish while other aspects like subcellular localization differ from the mammalian RSPO1.