A novel thermostable and organic solvent-tolerant lipase from <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> YB103: screening,purification and characterization

Thermostable lipases offer major biotechnological advantages over mesophilic lipases. In this study, an intracellular thermostable and organic solvent-tolerant lipase-producing strain YB103 was isolated from soil samples and identified taxonomically as Xanthomonas oryzae pv. oryzae. The lipase from X. oryzae pv. oryzae YB103 (LipXO) was purified 101.1-fold to homogeneity with a specific activity of 373.9 U/mg. The purified lipase showed excellent thermostability, exhibiting 51.1 % of its residual activity after incubation for 3 days at 70 °C. The enzyme showed optimal activity at 70 °C, suggesting it is a thermostable lipase. LipXO retained 75.1–154.1 % of its original activity after incubation in 20 % (v/v) hydrophobic organic solvents at 70 °C for 24 h. Furthermore, LipXO displayed excellent stereoselectivity (e.e._p >99 %) toward (S)-1-phenethyl alcohol in n-hexane. These unique properties of LipXO make it promising as a biocatalyst for industrial processes.     

<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in vitro mitogen or antigen-induced blastogenesis in man^2,3 and in animals (M. Bennett, E. G. Mayhew and T. Han, unpublished data) and suppresses bone-marrow derived antibody precursor cells in the mouse⁴.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.     

Vegetation of the <Emphasis Type="Italic">Hydrochari-Lemnete</Emphasis> and <Emphasis Type="Italic">Potametea</Emphasis> classes in the Danube-Tisza-Danube hydrosystem (Serbia)

Aquatic vegetation of Hydrochari-Lemnetea and Potametea classes in the Danube-Tisza-Danube hydrosystem (Hs DTD) was studied in 2009–2012, by applying the standard Braun-Blanquet method. The canal network vegetation comprises 14 associations, with Trapetum natantis and Ceratophylletum demersi being the most widely distributed. Hs DTD is also a habitat for several important endangered species, which serve as edificators of the following phytocenoses: Nymphaeetum albae, Nymphaeetum albo-luteae, Nymphoidetum peltatae, Trapetum natantis, Lemno-Spirodeletum, Salvinio-Spirodeletum polyrrhizae, Lemno-Utricularietum vulgaris, Potametum nodosi, Myriophyllo-Potametum and Najadetum marinae. In the studied vegetation, we also found an invasive phytocenosis Elodeetum canadensis that did not have an expanding tendency, and Ceratophyllo demersi-Vallisnerietum spiralis that had this tendency, which made monitoring its stands necessary. Physico-chemical analyses of water, conducted at localities in which the studied phytocenoses thrive, revealed that the development and distribution of most phytocenoses is closely linked with specific habitat conditions. Among the studied parameters, the most significant for the phytocenoses differentiation were: pH, alkalinity, COD-MnO₄, BOD₅, NO ₃ ^? , NO ₂ ^? , PO ₄ ^3? and the concentration of total phosphorus.     

Expression of sorghum gene <Emphasis Type="Italic">SbSGL</Emphasis> enhances grain length and weight in rice

Grain weight is a major determining factor of rice (Oryza sativa L.) yield and the comprehensive embodiment of grain length, width, and thickness. Here, we describe the molecular and functional characterization of SbSGL (Sorghum bicolor L. stress tolerance and grain length), a sorghum gene that encodes a putative member of the DUF1645 protein family of unknown function. Expression of SbSGL in rice promoted cell division and grain filling, which affected an array of traits of rice, including grain length, grain weight, and seed setting rate. Expression of SbSGL also affected the expression of genes related to the plant cell cycle and grain size.     

Combinatorial analysis of enzymatic bottlenecks of <Emphasis Type="SmallCaps">l</Emphasis>-tyrosine pathway by <Emphasis Type="Italic">p</Emphasis>-coumaric acid production in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Objective

To identify new enzymatic bottlenecks of l-tyrosine pathway for further improving the production of l-tyrosine and its derivatives.

Result

When ARO4 and ARO7 were deregulated by their feedback resistant derivatives in the host strains, the ARO2 and TYR1 genes, coding for chorismate synthase and prephenate dehydrogenase were further identified as new important rate-limiting steps. The yield of p-coumaric acid in the feedback-resistant strain overexpressing ARO2 or TYR1, was significantly increased from 6.4 to 16.2 and 15.3 mg l^?1, respectively. Subsequently, we improved the strain by combinatorial engineering of pathway genes increasing the yield of p-coumaric acid by 12.5-fold (from 1.7 to 21.3 mg l^?1) compared with the wild-type strain. Batch cultivations revealed that p-coumaric acid production was correlated with cell growth, and the formation of by-product acetate of the best producer NK-M6 increased to 31.1 mM whereas only 19.1 mM acetate was accumulated by the wild-type strain.

Conclusion

Combinatorial metabolic engineering provides a new strategy for further improvement of l-tyrosine or other metabolic biosynthesis pathways in S. cerevisiae.

     

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.     

Population structure analysis and association mapping of bacterial blight resistance in <Emphasis Type="Italic">indica</Emphasis> rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) accessions

Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is a serious disease in rice production worldwide. To understand the genetic diversity of bacterial blight resistance a population consisting of 175 indica accessions from nine countries was collected and detected their association between SSR (Simple Sequence Repeat) markers and resistance to six bacterial races. The resistance phenotypes of various rice accessions were evaluated through artificial inoculation under controlled conditions in 2013 and 2014. Association analysis showed that 17 SSR markers were significantly associated with resistance to four bacterial races and the phenotypic variations explained (PVE) ranged from 7.43 to 15.05%. Among the 17 associated SSR markers, two SSR markers located in previously reported genes regions, and 15 SSR markers were newly identified in this study. These results validated a new approach to map resistance genes of rice to bacterial blight. These markers could be used for marker-assisted selection (MAS) in rice bacterial blight resistance breeding programs.     

<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.     

Statistical techniques for modeling of <Emphasis Type="Italic">Corylus</Emphasis>, <Emphasis Type="Italic">Alnus</Emphasis>, and <Emphasis Type="Italic">Betula</Emphasis> pollen concentration in the air

Prediction of allergic pollen concentration is one of the most important goals of aerobiology. Past studies have used a broad range of modeling techniques; however, the results cannot be directly compared owing to the use of different datasets, validation methods, and evaluation metrics. The main aim of this study was to compare nine statistical modeling techniques using the same dataset. An additional goal was to assess the importance of predictors for the best model. Aerobiological data for Corylus, Alnus, and Betula pollen counts were obtained from nine cities in Poland and covered between five and 16 years of measurements. Meteorological data from the AGRI4CAST project were used as a predictor variables. The results of 243 final models (3 taxa \(\times\)  9 cities \(\times\) 9 techniques) were validated using a repeated k-fold cross-validation and compared using relative and absolute performance statistics. Afterward, the variable importance of predictors in the best models was calculated and compared. Simple models performed poorly. On the other hand, regression trees and rule-based models proved to be the most accurate for all of the taxa. Cumulative growing degree days proved to be the single most important predictor variable in the random forest models of Corylus, Alnus, and Betula. Finally, the study suggested potential improvements in aerobiological modeling, such as the application of robust cross-validation techniques and the use of gridded variables.     

Production of optically pure 2,3-butanediol from <Emphasis Type="Italic">Miscanthus floridulus</Emphasis> hydrolysate using engineered <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> strains

2,3-Butanediol (2,3-BD) can be produced by fermentation of natural resources like Miscanthus. Bacillus licheniformis mutants, WX-02ΔbudC and WX-02ΔgldA, were elucidated for the potential to use Miscanthus as a cost-effective biomass to produce optically pure 2,3-BD. Both WX-02ΔbudC and WX-02ΔgldA could efficiently use xylose as well as mixed sugars of glucose and xylose to produce optically pure 2,3-BD. Batch fermentation of M. floridulus hydrolysate could produce 21.6 g/L d-2,3-BD and 23.9 g/L meso-2,3-BD in flask, and 13.8 g/L d-2,3-BD and 13.2 g/L meso-2,3-BD in bioreactor for WX-02ΔbudC and WX-02ΔgldA, respectively. Further fed-batch fermentation of hydrolysate in bioreactor showed both of two strains could produce optically pure 2,3-BD, with 32.2 g/L d-2,3-BD for WX-02ΔbudC and 48.5 g/L meso-2,3-BD for WX-02ΔgldA, respectively. Collectively, WX-02ΔbudC and WX-02ΔgldA can efficiently produce optically pure 2,3-BD with M. floridulus hydrolysate, and these two strains are candidates for industrial production of optical purity of 2,3-BD with M. floridulus hydrolysate.     

<Emphasis Type="Italic">Drosophila melanogaster</Emphasis> as a polymicrobial infection model for <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> and <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Non-mammalian infection models have been developed over the last two decades, which is a historic milestone to understand the molecular basis of bacterial pathogenesis. They also provide small-scale research platforms for identification of virulence factors, screening for antibacterial hits, and evaluation of antibacterial efficacy. The fruit fly, Drosophila melanogaster is one of the model hosts for a variety of bacterial pathogens, in that the innate immunity pathways and tissue physiology are highly similar to those in mammals. We here present a relatively simple protocol to assess the key aspects of the polymicrobial interaction in vivo between the human opportunistic pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, which is based on the systemic infection by needle pricking at the dorsal thorax of the flies. After infection, fly survival and bacteremia over time for both P. aeruginosa and S. aureus within the infected flies can be monitored as a measure of polymicrobial virulence potential. The infection takes ~24 h including bacterial cultivation. Fly survival and bacteremia are assessed using the infected flies that are monitored up to ~60 h post-infection. These methods can be used to identify presumable as well as unexpected phenotypes during polymicrobial interaction between P. aeruginosa and S. aureus mutants, regarding bacterial pathogenesis and host immunity.     

Towards the mode of action of <Emphasis Type="Italic">Strobilanthes crispus</Emphasis> through integrated computational and experimental analyses

Bioactive sub-fractions from the tropical herbal plant Strobilanthes crispus (S. crispus) has been shown to induce apoptosis of breast cancer cells in vitro and reduce tumor size in vivo by our earlier studies. We have recently isolated five major compounds from S. crispus sub-fraction, namely lutein, β-sitosterol, campesterol, stigmasterol and pheophytin a. In this study, we set out to investigate each compound’s protein targets and mechanism of action through prediction of protein targets via a ligand-based target prediction protocol, Prediction IncluDinG INactives, and radioligand binding assays. The three phytosterol molecules (β-sitosterol, campesterol, stigmasterol) showed enrichment of hormone signaling GO terms [average ratio (AR) <0.01], while the SMAD signaling pathway was associated with pheophytin a (AR < 0.01). GO terms associated with retinoic acid receptor (RAR) and retinoid X receptor (RXR) were distinctly represented by protein targets of lutein (AR < 0.01). All members of the RAR/RXR family of proteins were predicted to be targeted by lutein, a feature that was not present in the other four S. crispus-derived compounds. Radioligand binding assay in vitro validated that lutein showed higher binding affinity with RXRα (IC₅₀: 5.74 µM; K_i: 4.55 µM) than RARα (IC₅₀: 25.1 µM; K_i: 14 µM). Molecular docking analysis demonstrated that lutein could occupy a large hydrophobic cavity of the hRXRα-LBP crystal structure mainly through hydrophobic interactions with leucine and isoleucine residues, and also hydrogen bond between a hydroxyl group of lutein with Glu²³⁹. Our findings suggest that lutein-RXRα interaction might play a role in the anti-breast cancer effects rendered by S. crispus.     

The <Emphasis Type="Italic">yajC</Emphasis> gene from <Emphasis Type="Italic">Lactobacillus buchneri</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> and its role in ethanol tolerance

The yajC gene (Lbuc_0921) from Lactobacillus buchneri NRRL B-30929 was identified from previous proteomics analyses in response to ethanol treatment. The YajC protein expression was increased by 15-fold in response to 10 % ethanol vs 0 % ethanol. The yajC gene encodes the smaller subunit of the preprotein translocase complex, which interacts with membrane protein SecD and SecF to coordinate protein transport and secretion across cytoplasmic membrane in Escherichia coli. The YajC protein was linked to sensitivity to growth temperatures in E. coli, involved in translocation of virulence factors during Listeria infection, and stimulating a T cell-mediated response of Brucella abortus. In this study, the L. buchneri yajC gene was over-expressed in E. coli. The strain carrying pET28byajC that produces YajC after isopropyl β-d-1-thiogalactopyranoside induction showed tolerance to 4 % ethanol in growth media, compared to the control carrying pET28b. This is the first report linking YajC to ethanol stress and tolerance.